RNA fragmentation assay

Abstract

Provided herein are methods of detecting fragmentation of an RNA transcript in a sample wherein the method comprises amplifying a first RNA sequence to a multi-partially fragmented RNA transcript using a first primer pair, and amplifying a second RNA sequence of the RNA transcript using a second primer pair, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment.

Description

1. BACKGROUND Traditional methods for detecting and characterizing RNA transcript (e.g., RNA-seq) fragmentation provide only an average measurement of RNA fragments in a population of cells, without quantifying differences in fragmentation patterns. Thus, there is a need for techniques that overcome these limitations and provide a more accurate, quantitative, and comprehensive understanding of RNA fragmentation.

[0002] 2. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said.xml copy, created on July 17, 2024, is named 35637-59375 WO and is 30,932 bytes in size. 3. SUMMARY The present disclosure describes methods and compositions for detecting fragmentation of RNA transcripts in a sample. Understanding the structure of RNA transcripts, including whether they are fragmented, is valuable for several reasons. First, the structure of an RNA molecule greatly influences its function within a cell. Full-length, unfragmented transcripts can be translated into functional proteins, while fragments can not encode a complete protein or can not be translated at all. Second, fragmentation can be a regulatory mechanism that cells use to control gene expression, either by destabilizing certain mRNAs to reduce their expression or by generating fragments with different functions, such as regulatory non-coding RNAs. Third, changes in mRNA fragmentation patterns can be indicative of various cellular conditions, including stress responses, diseases (e.g., cancer), or neurodegenerative conditions. Thus, a comprehensive understanding of RNA fragmentation can provide insight into cellular function, gene regulation, and disease mechanisms.

[0004] The methods and compositions described herein are capable of detecting fragmentation of RNA transcripts in RNA isolated from a single compartment (e.g., a cell) or from at least a first compartment (e.g., a cell) and a second compartment (e.g., an extracellular vesicle). The methods and compositions described herein are not limited to detecting fragmentation of RNA transcripts in a single environment (e.g., in a particular compartment or group of compartments). Rather, the methods and compositions described herein enable detection of fragmentation of RNA transcripts from any sample (or one or more compartments), where RNA transcript structure can be determined and used to design primers capable of detecting fragmentation.

[0005] In one embodiment, the compositions and methods described herein can be used to detect fragmentation of RNA transcripts in a first compartment (e.g., one cell or multiple cells) and a second compartment (e.g., one extracellular vesicle (EV) or multiple EVs). Extracellular vesicles that are naturally produced within cells are membrane-bound 50-300 nm particles that contain DNA, RNA, and proteins, as well as other bioactive molecules. EVs are released from a parent cell and are transferred to recipient cells, where they deliver their cargo, which can initiate important biological responses (e.g., cell proliferation, differentiation, or apoptosis). EVs thus play a role in intercellular communication, an important process that helps maintain the health of complex organisms (M. Colombo et al. Annual review of cell and developmental biology et al. , 30: 255-289 (2014); X. Zhang Frontiers in Cell and Developmental Biology , Figure 1A , vol. 9. Frontiers Media S.A., Nov. 05, 2021. doi: 10.3389 / fcell.2021.777441).

[0006] EVs produced in tissues are released from cells and enter the bloodstream. As such, liquid biopsies are an excellent source of EVs that can be monitored for patient health in clinical diagnostic tests. Cells grown in vitro also produce EVs and release them into the culture medium. EVs isolated from culture medium are a well-recognized model system for studying EV biology.

[0007] Current EV RNA studies have largely focused on miRNAs, which as a class have regulatory roles on biological pathways and can participate in intercellular communication. Many of these studies use miRNA-specific reagents for RNA isolation and RNA-Seq library preparation; this can limit the discovery of non-miRNA transcripts.

[0008] The present disclosure describes the study of EV RNA using RNA isolation and RNA-Seq library preparation reagents that assess the entire transcriptome from small miRNAs to large mRNAs and non-coding RNAs. This discovery shows that the EV transcriptome is very different from the cellular transcriptome, with key differences in the transcriptional architecture of many non-coding small RNAs. This work provides a “transcript fragmentation assessment PCR assay” that can distinguish EV RNA from cellular RNA based on transcript architecture differences using quantitative PCR and droplet digital PCR (ddPCR). Such an assay is able to quantify EV-specific transcripts that can ultimately be used as biomarkers for monitoring human health.

[0009] This disclosure includes a method for detecting fragmentation of RNA transcripts in a sample, the method comprising: adding a first primer pair and a second primer pair to the sample, wherein the sample comprises: a first RNA sequence of at most partially fragmented RNA transcripts; a second RNA sequence of the RNA transcripts, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment, the first primer pair being capable of hybridizing with the first RNA sequence and amplifying all or part of the first RNA sequence; the second primer pair being capable of hybridizing with the first RNA fragment and amplifying the first RNA fragment; amplifying the first RNA sequence and the first RNA fragment using the first and second primer pairs to produce a first amplification product comprising a sequence of the first RNA sequence or its reverse complementary sequence and a second amplification product comprising a sequence of the first RNA fragment or its reverse complementary sequence; quantifying the amount of the first amplification product and the amount of the second amplification product; and detecting fragmentation of RNA transcripts in the sample based on the amount of the second amplification product, wherein the amount of the second amplification product indicates RNA transcript fragmentation.

[0010] In some implementations, the first primer pair and the second primer pair are added to the sample in a separate reaction.

[0011] In some embodiments, the second RNA sequence is a fragment of the first RNA sequence. In some embodiments, the second primer set hybridizes with both the first RNA fragment of the second RNA sequence and a portion of the first RNA sequence. In some embodiments, the second primer set hybridizes with the first RNA fragment of the second RNA sequence but not with a portion of the first RNA sequence.

[0012] In some embodiments, the detection includes determining the amount of RNA transcript fragmentation in the sample based on the amount of the second amplification product, wherein the comparison between the amount of the second amplification product and the amount of the first amplification product indicates the amount or extent of RNA transcript fragmentation.

[0013] This disclosure includes a method for detecting fragmentation of RNA transcripts in a sample, the method comprising: adding at least a first primer pair, a second primer pair, and a third primer pair to the sample, wherein the sample comprises: a first RNA sequence of at most partially fragmented RNA transcripts; and a second RNA sequence of the RNA transcripts, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment, the first primer pair being capable of hybridizing with the first RNA sequence and amplifying all or part of the first RNA sequence; the second primer pair being capable of hybridizing with the first RNA fragment and amplifying the first RNA fragment; and the third primer pair being capable of hybridizing with the second RNA fragment and amplifying the second RNA fragment. Two RNA fragments; amplifying a first RNA sequence, a first RNA fragment, and a second RNA fragment using first, second, and third primer pairs, respectively, to produce a first amplification product containing the first RNA sequence or its reverse complementary sequence, a second amplification product containing the first RNA fragment or its reverse complementary sequence, and a third amplification product containing the second RNA fragment or its reverse complementary sequence; quantifying the amount of the first amplification product, the amount of the second amplification product, and the amount of the third amplification product; and detecting the fragmentation of the RNA transcript in the sample based on the average amount of the second amplification product and the third amplification product, wherein the average amount of the second amplification product and the third amplification product indicates RNA transcript fragmentation.

[0014] In some embodiments, the detection includes determining the amount of fragmented RNA transcript in the sample relative to the amount of the first amplification product based on the average amount of the second and third amplification products, wherein the comparison between the average amount of the second and third amplification products relative to the first amplification product indicates the amount of fragmented RNA transcript.

[0015] In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, or at least 10 nucleotides. In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, or at least 100 nucleotides.

[0016] In some implementations, the sample contains RNA from the first compartment, the second compartment, or both.

[0017] In some implementations, the first RNA sequence and the second RNA sequence are the same RNA transcript, but are present in both the first and second compartments.

[0018] In some implementations, the first compartment contains a first RNA sequence and a second RNA sequence, and / or the second compartment contains a first RNA sequence and a second RNA sequence.

[0019] In some embodiments, the method further includes determining the difference in the amount of RNA transcript fragmentation between the first and second compartments by measuring the amount or extent of a first or second RNA fragment of a first RNA sequence and a second RNA sequence in the first compartment and / or the second compartment. In some embodiments, determining the difference in the amount of RNA transcript fragmentation between the first and second compartments includes comparing the amount of RNA transcript fragmentation in the first compartment and the amount of RNA transcript fragmentation in the second compartment. In some embodiments, the method further includes one or more additional compartments. In some embodiments, the one or more additional compartments contain a first RNA sequence, a second RNA sequence, and / or a third RNA sequence.

[0020] In some embodiments, the first compartment contains RNA from multiple cells (e.g., cellular compartments), multiple extracellular vesicles (EVs) / exosomes (e.g., EV compartments), nuclear RNA (e.g., nuclear compartments), cytoplasmic RNA (e.g., cytoplasmic compartments), mitochondrial RNA (e.g., mitochondrial compartments), chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the first compartment, the second compartment, or both contain RNA from a control sample; or the first compartment contains RNA from a control sample comprising multiple cells, and the second compartment contains RNA from a suspected fragmented test sample, wherein the RNA from the test sample is selected from: multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some implementations, the first compartment, the second compartment, or both contain RNA from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples.

[0021] In some embodiments, the environmental sample is selected from water, wastewater, surface swabs, and air samples. In some embodiments, the sample contains RNA from multiple cells, RNA from multiple extracellular vesicles / exosomes, or both.

[0022] In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from an RNA transcript; and wherein the first and second RNA fragments of the second RNA sequence are fragments of the first RNA sequence. In some embodiments, all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both. In some embodiments, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment, the RNA transcript is considered to be expressed in both the first and second compartments. In some embodiments, the first compartment contains multiple cells, and the second compartment contains multiple extracellular vesicles / exosomes. In some embodiments, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes, the RNA transcript is considered to be expressed in both the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles. In some embodiments, the first RNA sequence is expressed in both the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles. In some embodiments, the amount of the first amplification product from the first compartment containing multiple cells is greater than the amount of the first amplification product from the second compartment containing multiple extracellular vesicles. In some embodiments, the first and second RNA fragments of the second RNA sequence are expressed in a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles. In some embodiments, the amount of the second amplification product from the second compartment containing multiple extracellular vesicles is higher than the amount of the first amplification product from the second compartment containing multiple extracellular vesicles.

[0023] In some embodiments, the first primer pair is capable of hybridizing and amplifying: a first RNA sequence from an RNA transcript comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes. In some embodiments, the second primer pair is capable of hybridizing and amplifying: a first RNA fragment from a second RNA sequence comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes. In some embodiments, the third primer pair is capable of hybridizing and amplifying: a second RNA fragment from a second RNA sequence comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes.

[0024] In some embodiments, the separation between the first and second RNA fragments in the second RNA sequence is reflected in the RNA sequencing data from the second compartment because the number of sequencing reads mapped to the nucleotides in the second RNA sequence that separate the first and second RNA fragments is reduced compared to the number of sequencing reads mapped to the corresponding nucleotides in the first RNA sequence in the first compartment. In some embodiments, the RNA transcript is non-coding RNA.

[0025] In some implementations, the non-coding RNA is selected from Y-RNA, snRNA, ncRNA, snoRNA, snaRNA, tRNA, rRNA, scRNA, telomerase RNA, fornix RNA, guide RNA, miRNA, antisense RNA, piRNA, and lncRNA.

[0026] In some embodiments, the method further includes selecting a first primer pair based on RNA sequencing data of a first RNA sequence from a first compartment. In some embodiments, RNA sequencing data of the first RNA sequence from the first compartment show that the first RNA sequence is fragmented in at most partially. In some embodiments, the method further includes selecting a second primer pair based on RNA sequencing data from a second compartment. In some embodiments, RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment. In some embodiments, the method further includes selecting a third primer pair based on RNA sequencing data from the second compartment. In some embodiments, the method further includes selecting at least a fourth primer pair, at least a fifth primer pair, at least a sixth primer pair, at least a seventh primer pair, at least an eighth primer pair, at least a ninth primer pair, or at least a tenth primer pair based on RNA sequencing data from the first compartment, the second compartment, or both.

[0027] In some implementations, RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment. In some implementations, the RNA sequencing data includes whole transcriptome RNA sequencing data, exome RNA sequencing data, targeted RNA sequencing data, small RNA sequencing, and miRNA sequencing.

[0028] In some embodiments, the method further includes isolating RNA from the sample, wherein the RNA comprises a first RNA sequence and a second RNA sequence. In the method of any one of claims 1-44, the second RNA sequence is fragmented in the sample prior to isolating the RNA from the sample. In some embodiments, the fragmentation of the second RNA sequence is not the result of sonication, enzymatic fragmentation, chemical modification, UV irradiation, or thermal degradation.

[0029] In some implementations, the fragmentation of the second RNA sequence is at least in part due to the presence of an exonuclease, an endonuclease, an RNA processing enzyme, an RNA-binding protein that binds to the second RNA sequence, or a combination thereof.

[0030] In some embodiments, the length of the first amplification product ranges from 50 to 220 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, or at least 70 nucleotides; or the second amplification product, the third amplification product, or both have a length of no more than 75 nucleotides, no more than 70 nucleotides, or no more than 67 nucleotides. In some embodiments, the length of the second amplification product, the third amplification product, or both ranges from 20 to 80 nucleotides.

[0031] In some embodiments, prior to the amplification, the method includes reverse transcription of the first RNA sequence and the first RNA fragment into complementary DNA (cDNA) in a polymerase chain reaction (PCR). In some embodiments, the first RNA sequence and the first RNA fragment are transcribed into complementary DNA (cDNA). In some embodiments, the first primer pair comprises: a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 and a reverse ... The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22.

[0032] According to any one of claims 1-53, the second primer pair comprises a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21.

[0033] In some embodiments, the primer in the first primer pair, the primer in the second primer pair, or the primer in the third primer pair contains one or more modified nucleotides. In some embodiments, the modified nucleotides are locked nucleotides.

[0034] One aspect of this disclosure includes a method for designing primers for detecting fragmentation of RNA transcripts, the method comprising: analyzing RNA sequencing data of RNA collected from a first compartment and RNA sequencing data of RNA collected from a second compartment, wherein the RNA sequencing data from the first compartment contains a first RNA sequence that is partially fragmented; the RNA sequencing data from the second compartment contains a first RNA sequence, wherein all or part of the first RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment; and the first RNA sequence, the first RNA fragment, and the second RNA fragment are present in both the first compartment and the second compartment; and selecting a first primer pair capable of hybridizing with the first RNA sequence and amplifying the first RNA sequence, and a second primer pair capable of hybridizing with the first RNA fragment.

[0035] One aspect of this disclosure includes a method for designing primers for detecting fragmentation of RNA transcripts, the method comprising: analyzing RNA sequencing data of RNA collected from a first compartment and RNA sequencing data of RNA collected from a second compartment; wherein the RNA sequencing data from the first compartment contains a first RNA sequence that is at most partially fragmented; the RNA sequencing data from the second compartment contains a first RNA sequence, wherein all or part of the first RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment; and the first RNA sequence, the first RNA fragment, and the second RNA fragment are present in both the first compartment and the second compartment; and selecting a first primer pair capable of hybridizing with and amplifying the first RNA sequence; a second primer pair capable of hybridizing with and amplifying the first RNA fragment; and a third primer pair capable of hybridizing with and amplifying the second RNA fragment.

[0036] In some embodiments, the first compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof. In some embodiments, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the first compartment, the second compartment, or both contain RNA from a control sample. In some embodiments, the first compartment, the second compartment, or both contain RNA from blood, plasma, liquid biopsy material, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples. In some embodiments, the environmental samples include water, wastewater, surface swabs, and air samples.

[0037] One aspect of this disclosure includes a method for detecting fragmentation of RNA transcripts in a sample, the method comprising: reverse transcribing the RNA transcripts into complementary DNA. (cDNA); Add a first primer pair and a second primer pair to the sample, wherein: the sample comprises: the inverse complementary sequence of a first RNA sequence of the RNA transcript, which is at most partially fragmented to produce the inverse complementary sequence of the first RNA sequence; the inverse complementary sequence of a second RNA sequence of the RNA transcript in the sample, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment; the first primer pair is capable of hybridizing with all or part of the inverse complementary sequence of the first RNA sequence and amplifying all or part of the inverse complementary sequence of the first RNA sequence; the second primer pair is capable of hybridizing with the inverse complementary sequence of the first RNA fragment and amplifying the inverse complementary sequence of the first RNA fragment; amplify the inverse complementary sequence of the first RNA sequence and the inverse complementary sequence of the first RNA fragment using the first and second primer pairs, thereby producing a first amplification product containing the inverse complementary sequence of the first RNA sequence and a second amplification product containing the inverse complementary sequence of the first RNA fragment; quantify the amount of the first amplification product and the amount of the second amplification product; and detect the fragmentation of the RNA transcript in the sample based on the ratio of the amount of the second amplification product to the amount of the first amplification product.

[0038] In some embodiments, the first primer pair and the second primer pair are added to the sample in a separate reaction. In some embodiments, after the first primer pair and the second primer pair are added to the sample but before the amplification, the method includes splitting the sample into multiple droplets.

[0039] In some embodiments, the method further includes diluting the cDNA before adding the first primer pair and the second primer pair to the sample. In some embodiments, the cDNA is diluted in water. In some embodiments, the dilution ratio is from 1:2 to 1:50.

[0040] In some embodiments, the method further includes: adding a third primer pair to the sample, wherein the third primer pair is capable of hybridizing and amplifying the second RNA fragment; using the third primer pair to amplify the second RNA fragment in a reaction different from the first RNA sequence and the first RNA fragment, thereby producing a third amplification product containing a sequence of the second RNA fragment or its reverse complementary sequence; quantifying the amount of the third amplification product; and detecting fragmentation of the RNA transcript in the sample based on one or more of the following: the ratio of the first amplification product, the second amplification product, and the third amplification product; the ratio of the first amplification product to the second amplification product; the ratio of the first amplification product to the average amount of the second and third amplification products; the ratio of the second amplification product to the first amplification product; the ratio of the second amplification product, the third amplification product, and the first amplification product; the ratio of the third amplification product to the first amplification product; and the ratio of the average amount of the second and third amplification products to the first amplification product.

[0041] In some embodiments, the sample is divided into multiple droplets comprising: a first set of droplets, each droplet containing the inverse complementary sequence (cDNA) of a first RNA sequence, a first primer pair, and one or more reagents; a second set of droplets, each droplet containing the inverse complementary sequence of a first RNA fragment, a second primer pair, and one or more reagents; optionally a third set of droplets, each droplet containing the inverse complementary sequence of a second RNA fragment, a third primer pair, and one or more reagents; and / or a subset of droplets that does not contain the inverse complementary sequence of the first RNA sequence, the inverse complementary sequence of the first RNA fragment, and / or the inverse complementary sequence of the second RNA fragment.

[0042] In some embodiments, the method further includes: subtracting the amount of the second amplicon product from the first amplicon product (second amplicon product – first amplicon product) and / or subtracting the amount of the third amplicon product from the first amplicon product (third amplicon product – first amplicon product). The first amplicon product is used to regulate the second and / or third amplicon products; and the fragmentation of the RNA transcript in the sample is detected based on one or more of the following: the ratio of a first amplicon product derived from multiple cells, a regulated second amplicon product derived from multiple cells, and a regulated third amplicon product derived from multiple cells; the ratio of a first amplicon product derived from multiple extracellular vesicles / exosomes, a regulated second amplicon product derived from multiple extracellular vesicles / exosomes, and a regulated third amplicon product derived from multiple extracellular vesicles / exosomes; the ratio of a first amplicon product derived from multiple cells to a regulated second amplicon product derived from multiple cells; the ratio of a first amplicon product derived from multiple extracellular vesicles / exosomes to a regulated second amplicon product derived from multiple extracellular vesicles / exosomes; the ratio of a first amplicon product derived from multiple cells to a regulated third amplicon product derived from multiple cells; the ratio of a first amplicon product derived from multiple cells to a regulated third amplicon product derived from multiple cells; the ratio of the average amount of a first amplicon product derived from multiple cells to a regulated second amplicon product derived from multiple cells; and the ratio of the average amount of a first amplicon product derived from multiple cells to a regulated second amplicon product derived from multiple cells; and the ratio of the average amount of a first amplicon product derived from multiple cells to a regulated second amplicon product derived from multiple cells; and the ratio of the average amount of a first amplicon product derived from multiple cells to a regulated third ... The ratio of the first amplified product of extracellular vesicles / exosomes to the average amount of regulated second amplified product and regulated third amplified product derived from multiple cells; the ratio of regulated second amplified product, regulated third amplified product, and first amplified product derived from multiple cells; the ratio of regulated second amplified product, regulated third amplified product, and first amplified product derived from multiple cells; the ratio of regulated second amplified product, regulated third amplified product, and first amplified product derived from multiple cells; the ratio of regulated second amplified product derived from multiple cells. The ratio of the product to the first amplification product derived from multiple cells; the ratio of the regulated second amplification product derived from multiple extracellular vesicles / exosomes to the first amplification product derived from multiple extracellular vesicles / exosomes; the ratio of the regulated third amplification product derived from multiple cells to the first amplification product derived from multiple cells; the ratio of the regulated third amplification product derived from multiple extracellular vesicles / exosomes to the first amplification product derived from multiple extracellular vesicles / exosomes; the average amount of the regulated second amplification product derived from multiple cells and the regulated third amplification product derived from multiple cells to the first amplification product derived from multiple cells;And the average amount of the regulated second amplification product and the regulated third amplification product derived from multiple extracellular vesicles / exosomes, relative to the first amplification product derived from multiple extracellular vesicles / exosomes.

[0043] In some embodiments, the first RNA sequence and the second RNA sequence originate from the same RNA transcript but are present in both the first and second compartments. In some embodiments, the first RNA fragment and the second RNA fragment are separated within the second RNA sequence by a spacer of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, or at least 10 nucleotides. In some embodiments, the first RNA fragment and the second RNA fragment are separated within the second RNA sequence by a spacer of at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, or at least 100 nucleotides. In some embodiments, the sample contains RNA from the first compartment, the second compartment, or both. In some embodiments, the method further includes determining the difference between the amount of fragmentation of RNA transcripts between the first compartment and the second compartment.

[0044] In some embodiments, determining the difference in the amount of RNA transcript fragmentation between the first and second compartments includes determining the ratio of the amount of RNA transcript fragmentation in the first compartment to the amount of RNA transcript fragmentation in the second compartment; or the ratio of the amount of RNA transcript fragmentation in the second compartment to the amount of RNA transcript fragmentation in the first compartment. In some embodiments, the method further includes one or more additional compartments. In some embodiments, the one or more additional compartments contain a first RNA sequence, a first RNA fragment, a second RNA fragment, or a third RNA sequence.

[0045] In some embodiments, the sample further comprises the inverse complementary sequence of a third RNA sequence of the RNA transcript, which is at most partially fragmented to produce the inverse complementary sequence of the third RNA sequence. In some embodiments, the sample further comprises the inverse complementary sequence of a fourth RNA sequence of the RNA transcript, which is at most partially fragmented to produce the inverse complementary sequence of the third RNA sequence. In some embodiments, the sample comprises the inverse complementary sequences of at least a fourth RNA sequence, at least a fifth RNA sequence, at least a sixth RNA sequence, at least a seventh RNA sequence, at least an eighth RNA sequence, at least a ninth RNA sequence, or at least a tenth RNA sequence.

[0046] In some embodiments, the first compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof. In some embodiments, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof. In some embodiments, the first compartment, the second compartment, or both contain RNA from a control sample containing multiple cells and a test sample containing multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0047] In some embodiments, the first compartment, the second compartment, or both contain RNA from blood, plasma, liquid biopsy material, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples. In some embodiments, the environmental samples are selected from water, wastewater, surface swabs, and air samples. In some embodiments, the sample contains RNA from multiple cells, RNA from multiple extracellular vesicles / exosomes, or both. In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from an RNA transcript or are the same RNA transcript. In some embodiments, all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

[0048] In some implementations, RNA transcripts are considered to be expressed in both the first and second compartments, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment.

[0049] In some embodiments, the first compartment contains multiple cells and the second compartment contains multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof, RNA transcripts are considered to be expressed in the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0050] In some implementations, the first RNA sequence is expressed in a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles.

[0051] In some embodiments, the amount of the first amplification product from a first compartment containing multiple cells is greater than the amount of the first amplification product from a second compartment containing multiple extracellular vesicles. In some embodiments, the first RNA fragment and the second RNA fragment of the second RNA sequence are expressed in both the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles. In some embodiments, the amount of the second amplification product from the second compartment containing multiple extracellular vesicles is higher than the amount of the first amplification product from the second compartment containing multiple extracellular vesicles. In some embodiments, the first primer pair is capable of hybridizing and amplifying: the first RNA sequence of the RNA transcript from the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles / exosomes. In some embodiments, the second primer pair is capable of hybridizing and amplifying: the first RNA fragment of the second RNA sequence from the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles / exosomes.

[0052] In some implementations, the ratio of the first compartment to the second compartment is determined using the following formula: Δcopy number （第一区室） =Amount of the second amplification product – Amount of the first amplification product; Δcopy number （第二区室） =Amount of the second amplification product – Amount of the first amplification product; X （第一区室） = ;as well as X （第二区室） = .

[0053] In some implementations, the detection of RNA transcript fragmentation in a sample is further based on a second ratio, wherein the second ratio is determined based on the following formula: Second ratio = Or the second ratio = .

[0054] In some implementations, the second ratio is not 1.

[0055] In some implementations, RNA transcripts are considered to be expressed in multiple cells and multiple extracellular vesicles, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes. In some implementations, the separation between the first and second RNA fragments in the second RNA sequence is reflected in the RNA sequencing data from the second compartment because the number of sequencing reads mapping to the nucleotides in the second RNA sequence that separate the first and second RNA fragments is reduced compared to the number of sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.

[0056] In some implementations, the RNA transcript is a non-coding RNA. In some implementations, the non-coding RNA is selected from γ-RNA, snRNA, ncRNA, snoRNA, snaRNA, tRNA, rRNA, scRNA, telomerase RNA, fornix RNA, guide RNA, miRNA, antisense RNA, piRNA, and lncRNA.

[0057] In some embodiments, the method further includes selecting a first primer pair based on RNA sequencing data from a first RNA sequence in a first compartment. In some embodiments, RNA sequencing data from the first RNA sequence in the first compartment show that the first RNA sequence is fragmented in at most partially. In some embodiments, the method further includes selecting a second primer pair based on RNA sequencing data from a second compartment. In some embodiments, RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment.

[0058] In some embodiments, the method further includes selecting a third primer pair based on RNA sequencing data from a second compartment. In some embodiments, the method further includes selecting at least a fourth, fifth, sixth, seventh, eighth, ninth, or tenth primer pair based on RNA sequencing data from a first, second, or both compartment.

[0059] In some embodiments, RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment. In some embodiments, the RNA sequencing data is whole transcriptome RNA sequencing data, exon RNA sequencing data, targeted RNA sequencing data, small RNA sequencing, and miRNA sequencing. In some embodiments, the method further includes isolating RNA from the sample, wherein the RNA comprises a first RNA sequence and a second RNA sequence. In some embodiments, the fragmentation of the second RNA sequence occurs in the sample prior to isolating the RNA from the sample.

[0060] In some embodiments, the fragmentation of the second RNA sequence is not the result of sonication, enzymatic fragmentation, chemical modification, UV irradiation, or thermal degradation. In some embodiments, the fragmentation of the second RNA sequence is at least in part due to the presence of exonucleases, endonucleases, RNA processing enzymes, RNA-binding proteins that bind to the second RNA sequence, or combinations thereof. In some embodiments, the length of the first amplification product ranges from 50 to 220 nucleotides.

[0061] In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 90 nucleotides, up to 80 nucleotides, up to 70 nucleotides, up to 60 nucleotides, up to 50 nucleotides, up to 40 nucleotides, up to 30 nucleotides, up to 20 nucleotides, or up to 10 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of at least 30 nucleotides. In some embodiments, the first primer pair comprises: a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 and a reverse ... The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22.

[0062] According to any one of claims 64-120, the second primer pair comprises: a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21.

[0063] In some embodiments, the primer in the first primer pair, the primer in the second primer pair, or the primer in the third primer pair contains one or more modified nucleotides. In some embodiments, the modified nucleotides are locked nucleotides.

[0064] One aspect of this disclosure includes a composition comprising a set of primers, comprising: a first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; and a second primer pair capable of hybridizing with and amplifying a first RNA fragment of a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0065] One aspect of this disclosure includes a composition comprising a set of primers, comprising: a first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; a second primer pair capable of hybridizing with and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair capable of hybridizing with and amplifying a second RNA fragment of a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0066] One aspect of this disclosure includes a composition comprising a set of primers, comprising: a first primer pair comprising a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 ... The sequence comprises: a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 15; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 15 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 16; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; a reverse The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10;A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21.

[0067] In some embodiments, both the primers in the first primer pair and the primers in the second primer pair contain one or more modified nucleotides. In some embodiments, the modified nucleotides are locked nucleotides.

[0068] One aspect of this disclosure includes a kit for determining the structure of an RNA transcript; the kit comprising: a first primer pair comprising a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; and a first primer pair comprising a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: ... having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; and a first primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 A forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 15; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11; The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10;The primers include: a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 21; and instructions for performing any method of this disclosure.

[0069] One aspect of this disclosure includes a kit for determining the structure of an RNA transcript; the kit comprising: a first primer pair comprising a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; and a first primer pair comprising a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: ... having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; and a first primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 A forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 15; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11; The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10;The primers include: a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 21; and instructions for performing any method of this disclosure. 4. Description of the attached drawings These and other features, aspects, and advantages of the invention will be better understood in conjunction with the following description and accompanying drawings, wherein: Figure 1B A flowchart is shown illustrating the steps for EV RNA isolation using qPCR to prepare an RNA Seq library for cell RNA isolation as described in Example 1.

[0071] Figure 1C A flowchart is shown illustrating the steps described in Example 1 for preparing an RNA-seq library for qPCR after isolating EVs and cellular RNA.

[0072] Figure 1D A flowchart is shown illustrating the RNA isolation steps commonly used in the preparation of samples for quantitative PCR (qPCR) and droplet digital PCR (ddPCR) as described in Example 1.

[0073] Figure 1E A flowchart is shown, illustrating the steps required for qPCR and ddPCR after SingleShot cell lysis.

[0074] Figure 1F A flowchart is shown, illustrating the steps required for qPCR after SingleShot cell lysis.

[0075] Figures 2A-2B A flowchart is shown, illustrating the steps required for ddPCR after SingleShot cell lysis.

[0076] Figure 2A The evaluation of the maximum RFU of the magnified trace is shown. Figure 2B ) and baseline thresholds at 25% and 75% of the maximum RFU (Figures 3A-3B ).

[0077] Figure 4 The analysis of the cellular RNA-Seq library ( Figure 4 A) or EV RNA-Seq library ( Figure 4 Example Bioanalyzer trace (B).

[0078] Figure 5A Visualization and analysis of target gene reads on the UCSC genome browser.

[0079] Figure 5A The transcript structure of RNY1 in cellular RNA and extracellular vesicle (EV) RNA is shown. Figure 5B The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNY1 gene on the UCSC Genome Browser. The RNY1 gene is 113 bp and is represented by blue bars. In the cellular RNA, the RNY1 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNY1 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNY1 transcript is fragmented into 5' and 3' fragments in the EV.

[0080] Figure 5B The transcript structure of RNY3 in cellular RNA and EV RNA is shown. Specifically, Figure 5C The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNY3 gene on the UCSC Genome Browser. The RNY3 gene is 102 bp and is represented by blue bars. In the cellular RNA, the RNY3 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNY3 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNY3 transcript is fragmented into 5' and 3' fragments in the EV.

[0081] Figure 5C The transcript structure of RNY4 in cellular RNA and EV RNA is shown. Figure 5DThe RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNY4 gene on the UCSC Genome Browser. The RNY4 gene is 96 bp and is represented by blue bars. In the cellular RNA, the RNY4 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNY4 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNY4 transcript is fragmented into 5' and 3' fragments in the EV.

[0082] Figure 5D (SEQ ID NO: 29) shows the transcript structure of RNY5 in cellular RNA and EV RNA assessed by transcript fragmentation PCR. Figure 5E The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNY5 gene on the UCSC Genome Browser. The RNY5 gene is 84 bp and is represented by blue bars. In the cellular RNA, the RNY5 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNY5 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNY5 transcript is fragmented into 5' and 3' fragments in the EV.

[0083] Figure 5E The transcript structure of RMRP in cellular RNA and EV RNA is shown. Figure 5F The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RMRP gene on the UCSC Genome Browser. The RMRP gene is 264 bp and is represented by blue bars. In cellular RNA, the RMRP transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in EV, the RMRP transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RMRP transcript is fragmented into 5' and 3' fragments in EV.

[0084] Figure 5F The transcript structure of RNU5B-1 in cellular RNA and EV RNA is shown. Figure 5GThe RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNU5B-1 gene on the UCSC Genome Browser. The RNU5B-1 gene is 116 bp and is represented by green bars. In the cellular RNA, the RNU5B-1 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNU5B-1 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNU5B-1 transcript is fragmented into 5' and 3' fragments in the EV.

[0085] Figure 5G The transcript structure of RNU5D-1 in cellular RNA and EV RNA is shown. Figure 5H The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNU5D-1 gene on the UCSC Genome Browser. The RNU5D-1 gene is 116 bp and is represented by green bars. In the cellular RNA, the RNU5D-1 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNU5D-1 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNU5D-1 transcript is fragmented into 5' and 3' fragments in the EV.

[0086] Figure 5H The transcript structure of RNU5E-1 in cellular RNA and EV RNA is shown. Figure 5I The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNU5E-1 gene on the UCSC Genome Browser. The RNU5E-1 gene is 120 bp and is represented by blue bars. In the cellular RNA, the RNU5E-1 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNU5E-1 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the RNU5E-1 transcript is fragmented into 5' and 3' fragments in the EV.

[0087] Figure 5I(SEQ ID NO: 30) shows the transcript structure of SNORA15 in cellular RNA and EV RNA. Figure 5J The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the SNORA15 gene on the UCSC Genome Browser. The SNORA15 gene is 133 bp and is represented by blue bars. In the cellular RNA, the SNORA15 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the SNORA15 transcript shows reads at the 5' and 3' ends, but fewer reads in the region between them. This data indicates that the SNORA15 transcript is fragmented into 5' and 3' fragments in the EV.

[0088] Figure 5J The transcript structure of RNU12 in cellular RNA and EV RNA is shown. Figure 5K The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in the red box) or EV RNA (last 6 tracks in the green box) from several cell lines, are visualized as custom tracks near the RNU12 gene on the UCSC Genome Browser. The RNU12 gene is 150 bp and is represented by blue bars. In the cellular RNA, the RNU12 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in the EV, the RNU12 transcript shows reads in the middle portion of the transcript, but significantly fewer reads at the 5' and 3' ends. This data suggests that the RNU12 transcript is fragmented in the EV and exists primarily as the middle portion of a gene lacking 5' and 3' ends.

[0089] Figure 5K The transcript structure of SCARNA10 in cellular RNA and EV RNA is shown. Figure 5LThe RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in red) or EV RNA (last 6 tracks in green) from several cell lines, are visualized as custom tracks near the SCARNA10 gene on the UCSC Genome Browser. The SCARNA10 gene is 330 bp and is represented by blue bars. In cellular RNA, the SCARNA10 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in EV, the SCARNA10 transcript shows reads in the middle portion of the transcript, but significantly fewer reads at the 5' and 3' ends. This data suggests that the SCARNA10 transcript is fragmented in EV and exists primarily as the middle portion of a gene lacking 5' and 3' ends.

[0090] Figure 5L The transcript structure of SCARNA5 in cellular RNA and EV RNA is shown. Figures 6A-6D The RNA-Seq results for the RNA samples, isolated from cellular RNA (first 6 tracks in red) or EV RNA (last 6 tracks in green) from several cell lines, are visualized as custom tracks near the SCARNA5 gene on the UCSC Genome Browser. The SCARNA5 gene is 276 bp and is represented by blue bars. In cellular RNA, the SCARNA5 transcript is intact because the reading pattern covers the entire coding region without breaks. Conversely, in EV, the SCARNA5 transcript shows reads in the middle portion of the transcript, but significantly fewer reads at the 5' and 3' ends. Furthermore, the middle portion of the SCARNA5 transcript is further fragmented by reads at the 5' and 3' ends, but fewer reads in the middle. This data indicates that in EV, the SCARNA5 transcript is fragmented and exists primarily as the middle portion of a gene lacking 5' and 3' ends. The middle portion is further fragmented into 5' and 3' fragments.

[0091] Figure 6A This shows the analysis of PCR for RNY5 by transcript fragmentation. Figure 6B (SEQ ID NO: 29) shows RNA-Seq data of cells and EV RNA isolated from several cell lines, revealing transcript fragmentation patterns of EV RNA. Figure 6C This shows the setup of primers for transcript fragmentation assessment PCR control and assay. Figure 6D (SEQ ID No: 1-4) shows the sequences of the PCR primers. Figures 7A-7DThe transcript fragmentation assessment PCR results showed that, in all cell lines, the RNY5 transcript was highly fragmented in EV RNA relative to cellular RNA with DDCq (which can be used interchangeably as “delta-delta Cq”, “DDCq”, or “ΔΔCq”) scores ranging from -7.41 to -12.84.

[0092] Figure 7A This shows the analysis of RNU12 by PCR evaluation through transcript fragmentation. Figure 7B This display shows RNA-Seq data of cells and EV RNA isolated from several cell lines, revealing transcript fragmentation patterns of the EV RNA. The height of the EV trajectory is customized to show details of the primer settings. Figure 7C This shows the setup of primers for transcript fragmentation assessment PCR control and assay. Figure 7D (SEQ ID No: 5-8) shows the sequence of the PCR primers. Figures 8A-8D Transcript fragmentation assessment PCR results showed that, in all cell lines, the RNU12 transcript was highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -5.40 to -12.42.

[0093] Figure 8A This shows the analysis of SCARNA5 by PCR assessment through transcript fragmentation. Figure 8B RNA-Seq data of cells and EV RNA isolated from several cell lines are shown, revealing the transcript fragmentation patterns of EV RNA. Figure 8C This shows the setup of primers for transcript fragmentation assessment PCR control and assay. Figure 8D (SEQ ID NO: 9-12) shows the sequence of the PCR primers. Figures 9A-9D The transcript fragmentation assessment PCR results showed that, in all cell lines, the SCARNA5 transcript was highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -5.96 to -8.75.

[0094] Figure 9A This shows the analysis of SCARNA10 by PCR evaluation through transcript fragmentation. Figure 9B RNA-Seq data from cells isolated from several cell lines and EV RNA revealed the transcriptional fragmentation patterns of EV RNA. Figure 9C This shows the setup of primers for transcript fragmentation assessment PCR control and assay. Figure 9D (SEQ ID No: 13-15) shows the sequence of the PCR primers. Figures 10A-10DThe transcript fragmentation assessment PCR results showed that, in all cell lines, the SCARNA10 transcript was highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -7.23 to -10.55.

[0095] Figure 10A This shows the analysis of RMRP by evaluating PCR through transcript fragmentation. Figure 10B RNA-Seq data of cells and EV RNA isolated from several cell lines are shown, revealing the transcript fragmentation patterns of EV RNA. Figure 10C This shows the setup of primers for transcript fragmentation assessment PCR control and assay. Figure 10D (SEQ ID No: 16-19) shows the sequence of the PCR primers. Figures 11A-11D The transcript fragmentation assessment PCR results showed that, in all cell lines except Jurkat, the RMRP transcript was highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -7.75 to -14.69.

[0096] Figure 11A This shows the analysis of RNY1 by evaluating PCR through transcript fragmentation. Figure 11B RNA-Seq data of cells and EV RNA isolated from several cell lines are shown, revealing the transcript fragmentation patterns of EV RNA. Figure 11C This shows the setup of primers for transcript fragmentation assessment PCR control and assay. Figure 11D (SEQ ID No: 20-22) shows the sequence of the PCR primers. Figures 12A-12B The transcript fragmentation assessment PCR results showed that, in all cell lines except Jurkat, the RNY1 transcript was fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -1.93 to -4.83.

[0097] Figure 12A The RNY5 EV fragment is shown, identified by Northern blotting and RNA-Seq. RNA Showing Chakrabortty et al. ( Figure 12B The figure (21 (11): 1966–1979 (2015), doi: 10.1261 / rna.053629.115) depicts the start (red) and end (blue) positions of RNY5 fragments isolated from EVs from BJ or K562 cells, as determined by Northern analysis. RNA(SEQ ID NO: 23) shows RNA-Seq data describing the structure of the RNY5 transcript found in Ntera-2EV. It is indicated according to Chakrabortty et al. ( Figures 13A-13E The start and end positions of RNY5 transcripts were determined by Northern blotting (21(11): 1966–1979 (2015), doi: 10.1261 / rna.053629.115). The structure of RNY5 transcripts in EVs is generally consistent. Note: Tracing molecular weights by Northern blotting is difficult and imprecise. The larger the size, the worse the inaccuracy. This can explain slight differences in transcript fragment positions.

[0098] Figure 13A The Y-RNA bases that interact with the Ro60 protein are shown. Xenopus laevis (SEQ ID NO: 24) shows the African clawed frog ( Cell The secondary structure of Y3 RNA, with the bases crucial for RO60 binding shown in pink (image courtesy of Stein et al.). Figures 13B-13E , 121(4): 529-539 (2005)). Figure 14 (SEQ ID Nos: 25-28) shows MCF7 exosomal RNA screenshots at loci RNY1, RNY3, RNY4, and RNY5. RNY1 is oriented from 3' to 5'. RNY3, RNY4, and RNY5 are oriented from 5' to 3'. The red box encloses the RO60-binding key base in the 5' region of the transcript. The blue box encloses the RO60-binding key base in the 3' region of the transcript. The yellow oval highlights the only non-conserved base in the sequence motif.

[0099] Figures 10A-10D Display usage Figure 15 The analysis of RMRP by transcript fragmentation using qPCR is shown as a comparison with the analysis of RMRP by transcript fragmentation using ddPCR. Transcript fragmentation using ddPCR is determined as follows: First, the “Δcopy number” is calculated, which is the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”), and then the cellular RNA sample (e.g., first compartment X) is calculated. (细胞RNA) ) and EV RNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA)The second ratio. ddPCR results in the A549 cell line showed that RMRP transcripts were highly fragmented in EV RNA relative to cellular RNA, similar to qPCR results.

[0100] Figures 6A-6D Display usage Figure 16 The analysis of RNY5 by transcript fragmentation using qPCR is shown in the comparison with the analysis of RNY5 by transcript fragmentation using ddPCR. Transcript fragmentation using ddPCR was determined as follows: First, the “Δcopy number” was calculated, which was the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”), and then the cellular RNA sample (e.g., first compartment X) was calculated. (细胞RNA) ) and EV RNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA) The second ratio. ddPCR results in the A549 cell line showed that, relative to cellular RNA, the RNY5 transcript was highly fragmented in EV RNA, similar to qPCR results.

[0101] Figures 7A-7D Display usage Figure 17 The analysis of RNU12 by qPCR assessment of transcript fragmentation shown is compared with the analysis of RNU12 by ddPCR assessment of transcript fragmentation. Transcript fragmentation using ddPCR was determined as follows: first, the “Δcopy number” was calculated, which was the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”), and then the cellular RNA sample (e.g., first compartment X) was calculated. (细胞RNA) ) and EV RNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA) The second ratio. ddPCR results in the A549 cell line showed that the RNU12 transcript was highly fragmented in EV RNA relative to cellular RNA, similar to qPCR results.

[0102] Figures 8A-8D Display usage Figure 18The analysis of SCARNA5 by qPCR assessment of transcript fragmentation shown is compared with the analysis of SCARNA5 by ddPCR assessment of transcript fragmentation. Transcript fragmentation using ddPCR was determined as follows: first, the “Δcopy number” was calculated, which was the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”), and then the cellular RNA sample (e.g., first compartment X) was calculated. (细胞RNA) ) and EV RNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA) The second ratio. ddPCR results in the A549 cell line showed that the SCARNA5 transcript was highly fragmented in EV RNA relative to cellular RNA, similar to qPCR results.

[0103] Figures 9A-9D Display usage Figure 19 The analysis of SCARNA10 by qPCR assessment of transcript fragmentation shown is compared with the analysis of SCARNA10 by ddPCR assessment of transcript fragmentation. Transcript fragmentation using ddPCR was determined as follows: first, the “Δcopy number” was calculated, which was the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”), and then the cellular RNA sample (e.g., first compartment X) was calculated. (细胞RNA) ) and EV RNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA) The second ratio. ddPCR results in the A549 cell line showed that the SCARNA10 transcript was highly fragmented in EV RNA relative to cellular RNA, similar to qPCR results.

[0104] Figures 11A-11D Display usage Figures 14-19 The analysis of RNY1 by qPCR assessment of transcript fragmentation shown is compared with the analysis of RNY1 by ddPCR assessment of transcript fragmentation. Transcript fragmentation using ddPCR was determined as follows: first, the “Δcopy number” was calculated, which was the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”), and then the cellular RNA sample (e.g., first compartment X) was calculated. (细胞RNA)) and EV RNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA) The second ratio. ddPCR results in the A549 cell line showed that, relative to cellular RNA, the RNY1 transcript was highly fragmented in EV RNA, similar to qPCR results. 5. Detailed Implementation 5.1. Methods for detecting RNA transcript fragmentation in samples This disclosure describes a method for detecting RNA transcript fragmentation in a sample, wherein the method includes using a first primer pair and a second primer pair. The method includes adding the first primer pair and the second primer pair to the sample. In some embodiments, the first primer pair and the second primer pair are added in different reactions.

[0106] In some embodiments, the sample comprises: a first RNA sequence of an RNA transcript that is at most partially fragmented (e.g., at most 40% (e.g., at most 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%)); and a second RNA sequence of the RNA transcript, wherein all or part of the second RNA sequence is fragmented into at least the first RNA fragment and optionally the second RNA fragment. In some embodiments, the first RNA sequence is not fragmented. A first primer pair is capable of hybridizing with the first RNA sequence and amplifying all or part of the first RNA sequence, and a second primer pair is capable of hybridizing with the first RNA fragment and amplifying the first RNA fragment.

[0107] In some embodiments, the method includes amplifying a first RNA sequence and a first RNA fragment using first and second primer pairs to produce a first amplification product comprising a sequence containing the first RNA sequence or its inverse complementary sequence, and a second amplification product comprising a sequence containing the first RNA fragment or its inverse complementary sequence. In some embodiments, the method further includes quantifying the amount of the first amplification product and the amount of the second amplification product; and detecting RNA transcript fragmentation in a sample based on the amount of the second amplification product, wherein the amount of the second amplification product relative to the first amplification product represents RNA transcript fragmentation.

[0108] In some implementations, "at most partially fragmented" means at most 40% fragmented. In some implementations, "at most partially fragmented" means at most 30% fragmented. In some implementations, "at most partially fragmented" means at most 20% fragmented. In some implementations, "at most partially fragmented" means at most 10% fragmented. In some implementations, "at most partially fragmented" means at most 5% fragmented. In some implementations, "at most partially fragmented" means at most 50% fragmented. In some implementations, "at most partially fragmented" means at most 60% fragmented. In some implementations, "at most partially fragmented" means at most 70% fragmented. In some implementations, "at most partially fragmented" means at most 80% fragmented. In some implementations, "at most partially fragmented" means at most 90% fragmented. In some implementations, the first RNA sequence is fragmented to a degree greater than 40%, 50%, 60%, 70%, 80%, or 90%. For example, in some implementations, a higher degree of fragmentation may occur if a large amount of the first RNA sequence exists in the cellular RNA compared to EV RNA.

[0109] In some implementations, the detection includes determining the amount of fragmented RNA transcripts in the sample based on the amount of the second amplification product, wherein the comparison between the amount of the second amplification product and the amount of the first amplification product serves as a test indicator of the amount of fragmented RNA transcripts.

[0110] This disclosure describes a method for detecting fragmentation of RNA transcripts in a sample, wherein the method includes using a first primer pair and a second primer pair. The method includes adding at least a first primer pair, a second primer pair, and a third primer pair to the sample. In some embodiments, at least a first primer pair, at least a second primer pair, and at least a third primer pair are added in different reactions. In some embodiments, the sample comprises: a first RNA sequence of at most partially fragmented RNA transcripts; and a second RNA sequence of RNA transcripts, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment. The first primer pair is capable of hybridizing with and amplifying all or part of the first RNA sequence, the second primer pair is capable of hybridizing with and amplifying the first RNA fragment, and the third primer pair is capable of hybridizing with and amplifying the second RNA fragment. The method includes amplifying a first RNA sequence, a first RNA fragment, and a second RNA fragment using first, second, and third primer pairs, respectively, to generate a first amplification product containing the first RNA sequence or its reverse complementary sequence, a second amplification product containing the first RNA fragment or its reverse complementary sequence, and a third amplification product containing the second RNA fragment or its reverse complementary sequence. The method further includes quantifying the amounts of the first amplification product, the second amplification product, and the third amplification product; and detecting RNA transcript fragmentation in the sample based on the average amount of the second and third amplification products, wherein the average amount of the second and third amplification products indicates RNA transcript fragmentation, and the amount of the first amplification product is used as a test.

[0111] In some implementations, the detection includes determining the amount of RNA transcript fragmentation in the sample based on the amount of the second amplification product, wherein a comparison between the amount of the second amplification product and the amount of the first amplification product indicates the amount of RNA transcript fragmentation.

[0112] In some embodiments, the sample contains RNA from a first compartment, a second compartment, or both. In some embodiments, the first compartment contains a first RNA sequence and the second compartment contains a second RNA sequence, or the first compartment contains a second RNA sequence and the second compartment contains a first RNA sequence, or the first compartment contains both a first RNA sequence and a second RNA sequence, or the second compartment contains both a first RNA sequence and a second RNA sequence.

[0113] 5.2. A method for detecting fragmentation of RNA transcripts in samples containing at least a first compartment and a second compartment. This disclosure describes a method for detecting fragmentation of RNA transcripts in a sample, wherein the method includes using a first primer pair and a second primer pair. The method includes adding the first primer pair and the second primer pair to the sample. The sample comprises: a first compartment containing a first RNA sequence of RNA transcripts, wherein the first RNA sequence is fragmented in at least a plurality of portions; and a second compartment containing a second RNA sequence of RNA transcripts, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment. The first primer pair is capable of hybridizing with the first RNA sequence and amplifying all or part of the first RNA sequence, and the second primer pair is capable of hybridizing with the first RNA fragment and amplifying the first RNA fragment. The method includes amplifying the first RNA sequence and the first RNA fragment using the first and second primer pairs, thereby producing a first amplification product containing a sequence of the first RNA sequence or its reverse complementary sequence, and a second amplification product containing a sequence of the first RNA fragment or its reverse complementary sequence. The method further includes quantifying the amount of the first amplification product and the amount of the second amplification product; and detecting RNA transcript fragmentation in the sample based on the amount of the second amplification product, wherein the amount of the second amplification product indicates RNA transcript fragmentation, and the amount of the first amplification product serves as the test, while the amount of the second amplification product serves as the control.

[0114] Compartmentation is a membrane-bound cellular organization that divides the internal volume of a cell into discrete (but usually interconnected) compartments. These compartments each have different properties and functions. For example, cellular compartments may include enclosed portions within the cytosol, surrounded by a single or double lipid membrane.

[0115] In some embodiments, the sample contains RNA from a first compartment, a second compartment, or both. In some embodiments, the first RNA sequence and the second RNA sequence are the same RNA transcript, but are present in both the first and second compartments. In some embodiments, the first compartment contains the first RNA sequence and the second compartment contains the second RNA sequence, or the first compartment contains the second RNA sequence and the second compartment contains the first RNA sequence. In some embodiments, the first compartment contains both the first and second RNA sequences. In some embodiments, the second compartment contains both the first and second RNA sequences.

[0116] In some embodiments, the method includes determining the difference in the amount of fragmentation of RNA transcript between the first compartment and the second compartment. In some embodiments, the first RNA sequence may be present in some compartments at a level similar to that of the second RNA fragment, such as a compartment containing multiple cells. In some embodiments, the first RNA sequence is present at a different level in the first compartment compared to the second compartment.

[0117] In some implementations, determining the difference between the amount of fragmentation of RNA transcripts in the first compartment and the second compartment involves comparing the amount of fragmentation of RNA transcripts in the first compartment with the amount of fragmentation of RNA transcripts in the second compartment.

[0118] In some embodiments, the first compartment contains RNA from multiple cells (e.g., cellular compartments), multiple extracellular vesicles (EVs) / exosomes (e.g., EV compartments), nuclear RNA (e.g., nuclear compartments), cytoplasmic RNA (e.g., cytoplasmic compartments), mitochondrial RNA (e.g., mitochondrial compartments), chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0119] In some embodiments, the first compartment, the second compartment, or both contain RNA from a control sample. In some embodiments, the first compartment contains RNA from a control sample comprising multiple cells, and the second compartment contains RNA from a test sample suspected of being fragmented, wherein the RNA from the test sample is selected from: multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0120] In some implementations, the first compartment, the second compartment, or both contain RNA from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples.

[0121] In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from an RNA transcript; and wherein the first and second RNA fragments of the second RNA sequence are fragments of the first RNA sequence.

[0122] In some implementations, all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

[0123] In some implementations, RNA transcripts are considered to be expressed in both the first and second compartments, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment.

[0124] In some implementations, the first compartment contains multiple cells and the second compartment contains multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum or combinations thereof.

[0125] In some implementations, RNA transcripts are considered to be expressed in both a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes.

[0126] In some implementations, the first RNA sequence is expressed in a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0127] In some embodiments, the amount of the first amplification product from a first compartment containing multiple cells is greater than the amount of the first amplification product from a second compartment containing multiple extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the amount of the first amplification product from a first compartment containing multiple cells is similar to the amount of the second amplification product from a first compartment containing multiple cells.

[0128] In some implementations, the first RNA fragment and the second RNA fragment in the second RNA sequence are expressed in a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles.

[0129] In some implementations, the amount of the second amplification product from the second compartment containing multiple extracellular vesicles is higher than the amount of the first amplification product from the second compartment containing multiple extracellular vesicles.

[0130] This disclosure includes a method for detecting fragmentation of RNA transcripts in a sample, wherein the method includes using a first primer pair and a second primer pair. The method includes adding at least a first primer pair, a second primer pair, and a third primer pair to the sample. The sample comprises: a first compartment containing a first RNA sequence, wherein the first RNA sequence of the RNA transcript is fragmented at most partially; and a second compartment containing a second RNA sequence of the RNA transcript, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment. The first primer pair is capable of hybridizing with and amplifying all or part of the first RNA sequence; the second primer pair is capable of hybridizing with and amplifying the first RNA fragment; and the third primer pair is capable of hybridizing with and amplifying the second RNA fragment. The method includes amplifying the first RNA sequence, the first RNA fragment, and the second RNA fragment using the first, second, and third primer pairs, respectively, to produce a first amplification product containing a sequence of the first RNA sequence or its reverse complementary sequence, a second amplification product containing a sequence of the first RNA fragment or its reverse complementary sequence, and a third amplification product containing a sequence of the second RNA fragment or its reverse complementary sequence. The method further includes quantifying: the amount of a first amplification product, the amount of a second amplification product, and the amount of a third amplification product; and detecting RNA transcript fragmentation in the sample based on the average amount of the second and third amplification products, wherein the average amount of the second and third amplification products indicates RNA transcript fragmentation, and the amount of the first amplification product is used as a test.

[0131] In some implementations, the sample comprises one or more additional compartments.

[0132] In some embodiments, the method includes isolating RNA from a first compartment, a second compartment, and one or more additional compartments. In some embodiments, one or more additional compartments contain a first RNA sequence, a first RNA fragment, and / or optionally a third RNA fragment.

[0133] In some implementations, the first primer pair is capable of hybridizing and amplifying the first RNA sequence from an RNA transcript comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes.

[0134] In some implementations, the second primer pair is capable of hybridizing and amplifying a first RNA fragment from a second RNA sequence comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes.

[0135] In some implementations, the third primer pair is capable of hybridizing and amplifying a second RNA fragment from a second RNA sequence comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes.

[0136] 5.3. RNA: Source, transcript, sequence In some embodiments, the sample comprises RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the sample comprises RNA from multiple cells (e.g., cell compartments), multiple extracellular vesicles (EVs) / exosomes (e.g., EV compartments), nuclear RNA (e.g., nuclear compartments), cytoplasmic RNA (e.g., cytoplasmic compartments), mitochondrial RNA (e.g., mitochondrial compartments), chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the sample comprises RNA from a compartment comprising an animal cell. In some embodiments, the sample comprises RNA from a eukaryotic cell compartment. In some embodiments, the sample comprises RNA from a plant cell compartment.

[0137] In some implementations, the sample contains RNA from multiple cells, RNA from multiple extracellular vesicles / exosomes, or both.

[0138] In some implementations, the sample contains RNA from blood, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant and / or environmental samples.

[0139] In some embodiments, the sample contains RNA from a first compartment, a second compartment, or both. In one embodiment, the first compartment includes a first RNA sequence and the second compartment includes a second RNA sequence, or the first compartment includes a second RNA sequence and the second compartment includes a first RNA sequence, or the first compartment includes both a first RNA sequence and a second RNA sequence, or the second compartment includes both a first RNA sequence and a second RNA sequence.

[0140] In some embodiments where the sample contains RNA from a first compartment and a second compartment, the first compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, and mitochondrial RNA. In some embodiments where the sample contains RNA from a first compartment and a second compartment, the first compartment contains RNA from multiple cells. In some embodiments where the sample contains RNA from a first compartment and a second compartment, the first compartment contains RNA from multiple extracellular vesicles / exosomes.

[0141] In some embodiments where the sample contains RNA from both the first and second compartments, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof.

[0142] In some embodiments where the sample comprises RNA from a first compartment and a second compartment, the first compartment comprises RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof; and the second compartment comprises RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof.

[0143] In some embodiments where the sample contains RNA from the first compartment and the second compartment, the first compartment, the second compartment, or both contain RNA from the control sample.

[0144] In some embodiments where the sample contains RNA from a first compartment and a second compartment, the first compartment, the second compartment, or both contain RNA from blood, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples. In such embodiments, the environmental sample is selected from water, wastewater, surface swabs, and air samples.

[0145] In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from an RNA transcript. In some embodiments, the first RNA sequence, the second RNA sequence, or both are the same RNA transcript. In some embodiments, the first RNA sequence, the second RNA sequence, or both are the same RNA transcript, but are present in both the first and second compartments.

[0146] In some embodiments where the sample contains RNA from the first compartment and the second compartment, all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

[0147] In some embodiments where the sample contains RNA from the first compartment and the second compartment, the RNA transcript is considered to be expressed in both the first compartment and the second compartment, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment.

[0148] In some embodiments where the sample contains RNA from a first compartment and a second compartment, the first compartment contains multiple cells and the second compartment contains multiple extracellular vesicles / exosomes. In such embodiments, RNA transcripts are considered to be expressed in both the multiple cells and the multiple extracellular vesicles, based in part on RNA sequencing data from the multiple cells and RNA sequencing data from the multiple extracellular vesicles / exosomes.

[0149] In some implementations, RNA transcripts are considered to be expressed in both a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes.

[0150] In some implementations, the RNA transcript is a non-coding RNA. In some implementations, the non-coding RNA is selected from: γ-RNA, snRNA, ncRNA, snoRNA, snaRNA, tRNA, rRNA, scRNA, telomerase RNA, fornix RNA, guide RNA, miRNA, antisense RNA, piRNA, and lncRNA.

[0151] In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, or at least 10 nucleotides.

[0152] In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, or at least 100 nucleotides.

[0153] In some implementations, the separation (gap) between the first and second RNA fragments in the second RNA sequence is reflected in the RNA sequencing data from the second compartment, with a reduced number of sequencing reads mapped to the second RNA sequence that separates the first and second RNA fragments compared to the number of sequencing reads mapped to the corresponding nucleotides in the first RNA sequence in the first compartment.

[0154] In some embodiments, the method includes selecting a first primer pair based on RNA sequencing data from a first RNA sequence in a first compartment. In some embodiments, RNA sequencing data from a first RNA sequence in a first compartment show that the first RNA sequence is fragmented at most partially. In this case, the fragmentation of the first RNA sequence is incorporated into the selection of the first primer pair.

[0155] In some embodiments, the method includes selecting a second primer pair based on RNA sequencing data from a second compartment. In some embodiments, RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment. In this case, the fragmentation of the second RNA sequence is incorporated into the selection of the second primer pair.

[0156] In some implementations, the method includes selecting a third primer pair based on RNA sequencing data from the second compartment. For example, RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment. In this case, the fragmentation of the second RNA sequence is incorporated into the selection of the third primer pair.

[0157] In some embodiments, the method includes selecting at least a fourth primer pair, at least a fifth primer pair, at least a sixth primer pair, at least a seventh primer pair, at least an eighth primer pair, at least a ninth primer pair, or at least a tenth primer pair based on RNA sequencing data from a first compartment, a second compartment, or both. In some embodiments, the method includes selecting more than two primer pairs, more than three primer pairs, more than four primer pairs, more than five primer pairs, more than six primer pairs, more than seven primer pairs, more than eight primer pairs, more than nine primer pairs, or more than ten primer pairs.

[0158] In some implementations, RNA sequencing data includes whole transcriptome RNA sequencing data, exon RNA sequencing data, targeted RNA sequencing data, small RNA sequencing, and miRNA sequencing. RNA sequencing data can be viewed on a genome viewer to visualize the location of sequencing reads within the RNA transcript sequence. A non-limiting example of a genome browser is the UCSC Genome Browser.

[0159] 5.4. Separation, (non-)fragmentation, and amplification In some embodiments, the method further includes isolating RNA from the sample, wherein the RNA comprises a first RNA sequence and a second RNA sequence.

[0160] In some embodiments, the method further includes isolating RNA from a sample, wherein the sample comprises a first compartment, a second compartment, or both, and RNA is isolated from each compartment separately. In some embodiments, the method further includes isolating RNA from a sample, wherein the sample comprises a first compartment, a second compartment, or both, and RNA is isolated from each compartment separately and then combined. In some embodiments, the method further includes isolating RNA from a sample, wherein the sample comprises a first compartment, a second compartment, or both, and RNA is isolated from both compartments in the same solution.

[0161] In some embodiments, the method includes culturing cells prior to RNA isolation. In some embodiments, the method includes precipitating EVs from suspended or adherent cells. In some embodiments, the method includes isolating EV RNA after precipitation. In some embodiments, the method includes isolating cellular RNA from suspended or adherent cells. In some embodiments, the method includes isolating cellular RNA after precipitation.

[0162] In some embodiments, a second RNA sequence is fragmented in the sample prior to RNA isolation. In some embodiments, the fragmentation of the second RNA sequence is not the result of sonication, enzymatic fragmentation, chemical modification, UV irradiation, and / or thermal degradation. In some embodiments, the method does not include a fragmentation step.

[0163] In some embodiments, the method includes preparing a sample comprising a first RNA sequence and a second RNA sequence isolated from an EV. In some embodiments, the method includes fragmentation of cellular RNA.

[0164] In some embodiments, the method includes end repair of EV and cellular RNA samples. In some embodiments, the method includes poly(A) tailing of EV and cellular RNA samples. In some embodiments, the method includes running EV and cellular RNA samples in a thermal cycler for continuous synthesis. In some embodiments, the method includes purifying and concentrating the inverse complementary sequence (cDNA) of EV and cellular RNA samples.

[0165] In some embodiments, amplification includes amplifying RNA transcripts (e.g., a first RNA sequence and a second RNA sequence) under conditions sufficient to allow amplification of the RNA transcripts. Amplification of the RNA transcripts (e.g., the first RNA sequence and the second RNA sequence) may include a reverse transcription step, whereby the RNA is reverse transcribed into complementary DNA (cDNA). Amplification of the RNA transcripts may include one or more of the following components: primers, which are short single-stranded DNA sequences complementary to a target sequence flanking region in the template DNA or template RNA; a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase), which is a thermostable DNA polymerase that synthesizes a new DNA strand using primers and template DNA or template RNA; nucleotides (dNTPs), for example, a mixture of deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, dGTP) to provide the A, T, C, and G bases for the polymerase to add to the new DNA strand; and a buffer solution providing conditions sufficient to enable the polymerase to function, wherein the buffer solution includes one or more salts, a pH buffer, and magnesium.

[0166] In some embodiments, the first amplification product has a length of at least 40 nucleotides (e.g., at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, at least 200 nucleotides, at least 210 nucleotides, at least 220 nucleotides, or at least 230 nucleotides). In some embodiments, the first amplification product has a nucleotide length of 40 to 230 nucleotide base pairs. In some embodiments, the first amplification product has a nucleotide length ranging from 50 to 220 nucleotide base pairs. In some embodiments, the first amplification product has a nucleotide length ranging from 55 to 215 nucleotide base pairs. In some embodiments, the length of the first amplification product does not exceed 220 nucleotides, or does not exceed 215 nucleotides.

[0167] In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 90 nucleotides, up to 80 nucleotides, up to 70 nucleotides, up to 60 nucleotides, up to 50 nucleotides, up to 40 nucleotides, up to 30 nucleotides, up to 20 nucleotides, or up to 10 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 90 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 80 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 70 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 60 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 50 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 40 nucleotides. In some embodiments, the second amplification product, the third amplification product, or both have a length of up to 30 nucleotides. In some implementations, the length of the second amplification product, the third amplification product, or both ranges from 20 to 80 nucleotides.

[0168] In some embodiments, the second amplification product, the third amplification product, or both are at least 20 nucleotides in length. In some embodiments, the first amplification product is at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 67 nucleotides, or at least 70 nucleotides in length. In some embodiments, the second amplification product, the third amplification product, or both are no more than 80 nucleotides or no more than 70 nucleotides in length.

[0169] In some embodiments, the method further includes determining the number of library amplification PCR cycles for each EV RNA and cell sample. For example, to determine the number of amplification cycles for the library, the method includes analyzing the first and second amplification products using two different thresholds, which effectively set upper and lower boundaries for cycles performing exponential amplification. In some embodiments, the method includes setting baseline thresholds of approximately 25% and 75% of the maximum RFU of the first amplicon product. In some embodiments, the method includes collecting Cq values ​​at 25% and 75% of the maximum RFU of the first and second amplicon products.

[0170] In some embodiments, the method includes analyzing RNA sequencing data to determine the structure of an RNA transcript (e.g., fragmentation). RNA sequencing data can be obtained from RNA isolated from an RNA sample obtained using the same methods described herein. For example, RNA can be isolated from multiple cells and used to obtain RNA sequencing data and to perform the methods described herein (e.g., transcript fragmentation assessment PCR). Alternatively, RNA sequencing data can be obtained from RNA isolated from an RNA sample obtained using methods different from those described herein.

[0171] RNA sequencing data can be generated from RNA sequence libraries prepared using RNA sequence library preparation kits. Non-limiting examples of kits that can be used to generate sequencing libraries include: Illumina TruSeq RNA Library Preparation Kit; NEBNext Ultra II Directional RNA Library Preparation Kit; QIAseq Stranded Total RNA Lib Preparation Kit; Takara SMART-Seq v4 Ultra Low Input RNA Kit; Lexogen QuantSeq 3' mRNA-Seq Library Preparation Kit; and KAPA Stranded mRNA-Seq Kit.

[0172] 5.5. Reverse transcription of RNA transcripts into complementary DNA (cDNA) In some embodiments, the RNA transcript of the sample is reverse transcribed into cDNA. In some embodiments, the first RNA sequence and the first RNA fragment are reverse transcribed into cDNA prior to amplification, depending on the PCR reaction performed (e.g., quantitative PCR or droplet digital PCR).

[0173] In some embodiments of qPCR, the method includes reverse transcribing a first RNA sequence into cDNA and performing qPCR and amplification in the same reaction. In some embodiments of qPCR, the method includes reverse transcribing a second RNA sequence (e.g., comprising a first RNA fragment and a second RNA fragment) into cDNA and performing qPCR and amplification in the same reaction. In some embodiments, the method includes performing cDNA transformation and qPCR in the same reaction. In some embodiments of qPCR, the method includes reverse transcribing a second RNA fragment into cDNA and performing qPCR in the same reaction. In some embodiments of qPCR, the method includes reverse transcribing a third RNA fragment into cDNA and performing qPCR in the same reaction. In some embodiments, a first primer pair and a second primer pair are added to the sample in separate reactions. In some embodiments, the method includes adding a first primer pair to a reaction containing the sample (e.g., a first reaction). In some embodiments, the method includes adding a second primer pair to a reaction containing the sample (e.g., a second reaction).

[0174] In some embodiments, when performing ddPCR, the method includes reverse transcribing the first RNA sequence into cDNA by mixing a first RNA sequence with a DNase master mixture and running the sample with the DNase master mixture in a thermal cycler, followed by mixing the sample with a reverse transcription master mixture and running the sample in a thermal cycler using an appropriate protocol. The resulting cDNA sample is then mixed with an EvaGreen Supermix. Therefore, in some embodiments, the method includes mixing the cDNA sample with an EvaGreen Supermix kit. In some embodiments, the method further includes generating droplets from the sample. After droplet generation, the method includes running the sample with the EvaGreen Supermix in a thermal cycler. In some embodiments, when ddPCR is used for amplification, the cDNA is prepared in batches.

[0175] 5.6. Primers, primer pairs, and primer sets This disclosure also describes primers, primer pairs, and primer pair sets that can be used to detect fragmentation of RNA transcripts in a sample.

[0176] In one implementation, the first primer pair is capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented at most partially.

[0177] In another embodiment, the second primer pair is capable of hybridizing with and amplifying the first RNA fragment of the second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least the first RNA fragment and the second RNA fragment.

[0178] In one embodiment, the primer pair set comprises: a first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; and a second primer pair capable of hybridizing with and amplifying a first RNA fragment of a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0179] In some embodiments, the method includes a first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; and a second primer pair capable of hybridizing with a first RNA fragment of a second RNA sequence and amplifying a first RNA fragment of the second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0180] In some embodiments, a first primer pair is capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; a second primer pair is capable of hybridizing with and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair is capable of hybridizing with and amplifying a second RNA fragment of a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0181] In some embodiments, the first primer pair is selected from: a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 and a reverse ... The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; and the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22.

[0182] In some embodiments, the second primer pair is selected from: a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse ... The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; and the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21.

[0183] In some embodiments, the primer pair set includes a first primer pair selected from: a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10 and a reverse ... The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; and the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22; and a second primer pair selected from the following: the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16; the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 Forward primers of sequences having at least 80% sequence identity with the sequence of sequence 3 and reverse primers of sequences having at least 80% sequence identity with the sequence of sequence 4; forward primers of sequences having at least 80% sequence identity with the sequence of sequence 6 and reverse primers of sequences having at least 80% sequence identity with the sequence of sequence 8; forward primers of sequences having at least 80% sequence identity with the sequence of sequence 9 and reverse primers of sequences having at least 80% sequence identity with the sequence of sequence 10;The sequence comprises: a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; and a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 21.

[0184] Designing PCR primers that target (and amplify) fragmented transcripts found in compartments (e.g., EVs) is challenging because many fragments are small (less than 30 bases) and cannot accommodate forward and reverse PCR primers.

[0185] In some embodiments, PCR primers include modifications that increase primer Tm and improve PCR performance. Non-limiting examples of these types of modifications include incorporation of one or more modified oligonucleotides (e.g., locked nucleic acids (LNAs), peptide nucleic acids (PNAs)) and incorporation of one or more modified internucleotide bonds (e.g., phosphate thioester bonds).

[0186] In some implementations, PCR primers include modifications that enhance their ability to target (and amplify) fragments (e.g., short sequences). Non-limiting examples of these types of modifications include incorporation of one or more modified oligonucleotides (e.g., locked nucleic acids (LNAs), peptide nucleic acids (PNAs)) and incorporation of one or more modified internucleotide bonds (e.g., phosphate thioester bonds).

[0187] In some cases, modified oligonucleotides can be incorporated into PCR primers to amplify fragmented transcripts that are otherwise difficult to amplify. Non-limiting examples of modified oligonucleotides include locked nucleic acids (LNAs), peptide nucleic acids (PNAs), oligonucleotides with non-natural bases (e.g., inosine), cytosine analogs (e.g., 5-methylcytosine), thymine analogs (5-bromouracil), adenine analogs (e.g., 2-aminoadenine), and guanine analogs.

[0188] In some implementations, locked nucleic acid bases are incorporated into PCR primers or PCR primer pairs to increase primer Tm and improve PCR performance.

[0189] 5.7. Determining the amount of fragmentation in RNA transcripts 5.7.1. Quantitative PCR (qPCR) In some embodiments, the method includes quantifying the amount of a first amplification product and the amount of a second amplification product. In some embodiments, quantifying the amount of the first amplification product and the amount of the second amplification product is performed using quantitative PCR. In quantitative PCR (qPCR), Δq is the quantitative cycle number (Cq). Cq is a calculation used to quantify the relative expression of the target gene compared to a reference or "housekeeping" gene. Cq is the cycle number at which fluorescence exceeds a threshold, typically set in the exponential phase of the amplification curve. ΔCq is calculated by subtracting the Cq value of the housekeeping gene from the Cq value of the target gene in the same sample. Cq = Cq (target gene) - Cq (reference gene). Lower Cq values ​​indicate high expression levels of the target gene, and higher Cq values ​​indicate higher expression levels of the target gene. Cq values ​​indicate low target gene expression levels. Normalization using housekeeping genes helps to account for variations in the quantity or quality of starting material, amplification efficiency, reverse transcription efficiency, and differences in total transcriptional activity between different samples or between different compartments (e.g., between the first and second compartments).

[0190] In some implementations, results (e.g., the amount of RNA) are normalized to determine if changes in Cq values ​​are due to genuine biological changes rather than technical issues. Non-limiting examples of normalization methods include “Δ-Cq,” “Δ-Δ-Cq,” the Pfaffl method, or the Livak method. In these examples, the Cq values ​​of a sample are compared to the Cq values ​​of several reference (housekeeper) genes.

[0191] The Δ-ΔCq method assumes that the amplification (PCR) efficiencies of the reference and target samples are nearly 100% and within 5% of each other. The Livak method, used for relative quantification, assumes that the PCR efficiencies of the target and reference genes should be between 90% and 100%. The Pfaffl method assumes that the reaction efficiencies of the reference and target genes differ.

[0192] Choosing a reference gene whose expression level is expected to remain unchanged during the experiment is a consideration for obtaining reliable and accurate results. Non-limiting examples of housekeeping genes include actin, α-tubulin, GAPDH, and ubiquitin.

[0193] It is worth noting that the Δ-ΔCq method assumes that the amplification (PCR) efficiencies of the reference and target samples are nearly 100% and within 5% of each other. Other normalization methods include the Δ-Cq method and the Pfaffl method.

[0194] In some embodiments, the method includes detecting RNA transcript fragmentation in a sample based on the amount of a second amplification product, wherein the amount of the second amplification product indicates RNA transcript fragmentation and the amount of the first amplification product serves as a control. In one embodiment, the method includes detecting fragmentation based on the amount of the second amplification product (… Cq) detects the fragmentation of RNA transcripts in a sample, where the amount of the second amplification product indicates RNA transcript fragmentation and the amount of the first amplification product ( Cq) was used as a test.

[0195] In some embodiments, the method includes detection comprising determining the amount of RNA transcript fragmentation in a sample based on the average amount of a second and a third amplification product, wherein a comparison between the average amount of the second and third amplification products and the amount of a first amplification product being tested indicates the amount of RNA transcript fragmentation. For example, the method includes detection comprising determining the amount of fragmentation of RNA transcript based on the average amount of the second and third amplification products (…). Cq) measures the amount of fragmented RNA transcripts in the sample ( Cq), where the average amount of the second and third amplification products (Cq) Cq) and the amount of the first amplification product used for testing ( Comparisons between Cq) indicate the amount of RNA transcript fragmentation.

[0196] In some embodiments, the method includes determining the difference between the amount of RNA transcript fragmentation in the first compartment and the second compartment. In some embodiments, determining the difference between the amount of RNA transcript fragmentation in the first compartment and the second compartment includes comparing the amount of RNA transcript fragmentation in the first compartment and the amount of RNA transcript fragmentation in the second compartment.

[0197] 5.7.1. Droplet Digital PCR (ddPCR) This disclosure includes methods for detecting fragmentation of RNA transcripts in samples using ddPCR. The method includes: reverse transcribing an RNA transcript into complementary DNA (cDNA); adding a first primer pair and a second primer pair to a sample, wherein: the sample contains: an inverse complementary sequence of a first RNA sequence of the RNA transcript, which is at most partially fragmented to produce an inverse complementary sequence of the first RNA sequence; an inverse complementary sequence of a second RNA sequence of the RNA transcript in the sample, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and optionally a second RNA fragment; the first primer pair is capable of hybridizing with the first RNA sequence and amplifying all or part of the inverse complementary sequence of the first RNA sequence; the second primer pair is capable of hybridizing with the inverse complementary sequence of the first RNA fragment and amplifying the inverse complementary sequence of the first RNA fragment; amplifying the inverse complementary sequence of the first RNA sequence and the inverse complementary sequence of the first RNA fragment using the first and second primer pairs, thereby producing a first amplification product containing the inverse complementary sequence of the first RNA sequence and a second amplification product containing the inverse complementary sequence of the first RNA fragment; quantifying the amount of the first amplification product and the amount of the second amplification product; and detecting the fragmentation of the RNA transcript in the sample based on the ratio of the amount of the second amplification product to the amount of the first amplification product.

[0198] In some embodiments, the first primer pair and the second primer pair are added in different reactions. In some embodiments, the test sample and the control sample can be included in the same reaction containing either the first primer pair or the second primer pair. In some embodiments, the first compartment and the second compartment can be included in the same reaction containing either the first primer pair or the second primer pair. In some embodiments, the test sample and the control sample can be included in different reactions. In some embodiments, the test sample contains multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, the control sample contains RNA from multiple cells. In some embodiments, the control sample... In some embodiments, the method includes quantifying the amount of a first amplification product and a second amplification product. In some embodiments, droplet digital PCR (ddPCR) is used to quantify the amount of the first amplification product and the second amplification product.

[0199] First, once cDNA is synthesized from the first RNA sequence and a second RNA sequence containing at least the first RNA fragment, a 20-22 μL reaction mixture is prepared using the cDNA of the first RNA sequence, and / or a second RNA sequence containing the first RNA fragment and optionally the second RNA fragment, reagents for detecting fragmentation of the RNA transcript, and primers. In some embodiments, the first RNA fragment, the second RNA fragment, and the first RNA sequence are reacted with their respective reagents and primers.

[0200] In some embodiments, when the sample is a cellular sample (e.g., cellular RNA reverse transcribed into cDNA), the cDNA is diluted prior to ddPCR. In some embodiments, the cDNA is diluted in water. In some embodiments, the dilution range is 1:2 to 1:50. In some embodiments, the dilution range is 1:2 to 1:500, 1:2 to 1:400, 1:2 to 1:300, 1:2 to 1:200, 1:2 to 1:100, or 1:2 to 1:50.

[0201] Next, the method includes generating a plurality of droplets. In some embodiments, for a 20 μL reaction, the plurality of droplets ranges from at least 15,000 droplets, at least 16,000 droplets, at least 17,000 droplets, at least 18,000 droplets, at least 19,000 droplets, or at least 20,000 droplets. In some embodiments, the plurality of droplets ranges from 16,000 to 20,000 droplets. In some embodiments, the plurality of droplets ranges from 18,000 to 20,000 droplets. In some embodiments, once the reaction mixture is ready, the method includes loading 20-22 μL of the reaction mixture into the sample well of the DG8 chamber for the QX200 droplet generator (Bio-Rad #1864008) according to the instructions in the automatic droplet generator instruction manual (Bio-Rad #10043138), and then loading 70 μL of QX200 Droplet Generation Oil for EvaGreen® (Bio-Rad #1864005 or 1864006) into the oil well. In some implementations, the sample is divided into multiple droplets, including: a first set of droplets, each droplet containing the reverse complementary sequence of a first RNA sequence (cDNA), a first primer pair, and one or more reagents; a second set of droplets, each droplet containing the reverse complementary sequence of a first RNA fragment, a second primer pair, and one or more reagents; optionally a third set of droplets, each droplet containing the reverse complementary sequence of a second RNA fragment, a third primer pair, and one or more reagents; and / or a subset of droplets that does not contain the reverse complementary sequence of the first RNA sequence, the reverse complementary sequence of the first RNA fragment, and / or the reverse complementary sequence of the second RNA fragment.

[0202] In some implementations, the method further includes DNA fragmentation via restriction digestion prior to droplet generation. Restriction enzyme digestion can be performed as a separate reaction prior to the ddPCR reaction setup. After generating droplets using the QX200 droplet generator according to the manufacturer's instructions, the method includes running the sample in a thermal cycler, followed by reading the droplets in the QX200 droplet reader (Bio-Rad) according to the manufacturer's instructions. Following thermal cycling, the method includes analyzing the sample using QX Manager software. The QX Manager software performs data acquisition and analyzes the droplets using EvaGreen fluorescent dye. The concentration of the amplicon product (e.g., first amplicon product, second amplicon product, or both) is reported as copies / μL of the final 1x ddPCR reaction.

[0203] In some embodiments, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the first, second, and third amplification products. In some embodiments, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the first and second amplification products. In some embodiments, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the first amplification product to the average amount of the second and third amplification products. In some embodiments, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the second amplification product to the first amplification product. In some embodiments, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the second, third, and first amplification products. In some embodiments, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the third amplification product to the first amplification product. In some implementations, when using ddPCR, fragmentation of RNA transcripts in a sample is detected based on the ratio of the average amount of the second and third amplification products to the first amplification product.

[0204] In some implementations, before detecting the ratio of the second amplification product to the first amplification product; the ratio of the third amplification product to the first amplification product, or the average of the second and third amplification products to the first amplification product, the method includes calculating the "" of the second RNA sequence. The copy number is adjusted or calculated based on the presence of the first amplification product to determine the second or third amplification product. The copy number of the second RNA sequence... "Copy number" is, for example, the amount of a second or third amplified product from a second RNA sequence minus the amount of a first amplified product from a first RNA sequence. For example, as... Figures 14-19 As shown, the second RNA sequence " The "copy number" is displayed as the amount of the second amplified product minus the amount of the first amplified product (e.g., "average control" – "average test"). In some embodiments, the method further includes calculating the "copy number" of the cellular RNA sample (e.g., the first compartment) and the EVRNA sample (e.g., the second compartment). The first ratio (X) of "copy number" and "average trial", and X (细胞RNA) With X (EV RNA) The second ratio.

[0205] In some implementations, fragmentation of RNA transcripts in a sample is detected based on one or more of the following: the ratio of a first amplification product from multiple cells, a second amplification product from multiple cells, and a third amplification product from multiple cells; the ratio of a first amplification product from multiple extracellular vesicles / exosomes, a second amplification product from multiple extracellular vesicles / exosomes, and a third amplification product from multiple extracellular vesicles / exosomes; the ratio of a first amplification product from multiple cells to a second amplification product from multiple cells; the ratio of a first amplification product from multiple extracellular vesicles / exosomes to a second amplification product from multiple extracellular vesicles / exosomes; the ratio of a first amplification product from multiple cells to a third amplification product from multiple cells; the ratio of a first amplification product from multiple extracellular vesicles / exosomes to a third amplification product from multiple extracellular vesicles / exosomes; the ratio of the average amount of a first amplification product from multiple cells to a second amplification product from multiple cells and a third amplification product from multiple cells; and the ratio of a first amplification product from multiple extracellular vesicles / exosomes to a second amplification product from multiple cells and a third amplification product from multiple extracellular vesicles / exosomes. The ratio of the average amount of the third amplification product; the ratio of the second amplification product, the third amplification product, and the first amplification product from multiple cells; the ratio of the second amplification product, the third amplification product, and the first amplification product from multiple extracellular vesicles / exosomes; the ratio of the second amplification product from multiple cells to the first amplification product from multiple cells; the ratio of the second amplification product from multiple cells to the first amplification product from multiple cells; the ratio of the third amplification product from multiple cells to the first amplification product from multiple cells; the ratio of the third amplification product from multiple cells to the first amplification product from multiple cells; the ratio of the average amount of the second amplification product and the third amplification product from multiple cells to the first amplification product from multiple cells; and the ratio of the average amount of the second amplification product and the third amplification product from multiple cells to the first amplification product from multiple cells.

[0206] In some implementations, the first RNA sequence and the second RNA sequence are derived from the same RNA transcript, but are present in both the first and second compartments.

[0207] In some implementations, the sample contains RNA from the first compartment, the second compartment, or both.

[0208] In some embodiments, the method further includes determining the difference between the amount of fragmented RNA transcripts between the first compartment and the second compartment. In some embodiments, determining the difference between the amount of fragmented RNA transcripts between the first compartment and the second compartment includes determining the ratio of the amount of fragmented RNA transcripts in the first compartment to the amount of fragmented RNA transcripts in the second compartment; or the ratio of the amount of fragmented RNA transcripts in the second compartment to the amount of fragmented RNA transcripts in the first compartment.

[0209] In some embodiments, the first compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof. In some embodiments, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or combinations thereof. In some embodiments, the first compartment, the second compartment, or both contain RNA from a control sample and a test sample, wherein the control sample comprises multiple cells and the test sample comprises multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0210] The RNA transcript is expressed in all or part of the first compartment, the second compartment, or both.

[0211] In some implementations, RNA transcripts are considered to be expressed in both the first and second compartments, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment.

[0212] In some embodiments, the first compartment contains multiple cells, and the second compartment contains multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof. In some embodiments, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof, RNA transcripts are considered to be expressed in the first compartment containing multiple cells and the second compartment containing multiple extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0213] In some implementations, the first RNA sequence is expressed in a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles.

[0214] In some implementations, the amount of the first amplification product from a first compartment containing multiple cells is greater than the amount of the first amplification product from a second compartment containing multiple extracellular vesicles.

[0215] In some implementations, the first RNA fragment and the second RNA fragment in the second RNA sequence are expressed in a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles.

[0216] In some implementations, the amount of the second amplification product from the second compartment containing multiple extracellular vesicles is higher than the amount of the first amplification product from the second compartment containing multiple extracellular vesicles.

[0217] In some embodiments, the first primer pair is capable of hybridizing and amplifying: a first RNA sequence from an RNA transcript comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes. In some embodiments, the second primer pair is capable of hybridizing and amplifying: a first RNA fragment from a second RNA sequence comprising a first compartment containing multiple cells and a second compartment containing multiple extracellular vesicles / exosomes.

[0218] In some implementations, when using ddPCR, the detection of RNA transcript fragmentation is determined using the ratio of the first and second compartments according to the following formula: Δcopy number （第一区室） =Amount of the second amplification product – Amount of the first amplification product; Δcopy number （第二区室） =Amount of the second amplification product – Amount of the first amplification product; X （第一区室） = ;as well as X （第二区室） = .

[0219] In some implementations, the detection of RNA transcript fragmentation in a sample is further based on a second ratio, wherein the second ratio is determined based on the following formula: Second ratio = .

[0220] In some implementations, the detection of RNA transcript fragmentation in a sample is further based on a second ratio, wherein the second ratio is determined based on the following formula: Second ratio == .

[0221] In some implementations, the second ratio is not 1. For example, such as... Figure 2AAs shown, transcript fragmentation using ddPCR is determined by first adjusting the second or third amplification product in response to the presence of the first amplification product (e.g., in the case where the second primer pair hybridizes with a first RNA fragment and a portion of the first RNA sequence). For example, in the case where the second primer set amplifies a first RNA fragment and a portion of the first RNA sequence, the second and third amplification products are “corrected” by the first amplification product (subtracting the amount of the first transcript from the amount of the second transcript). In some embodiments, adjusting the second amplification product includes subtracting the amount of the second amplification product from the amount of the first amplification product from the first RNA sequence to determine the “Δ copy number.” In some embodiments, the method further includes, after calculating the Δ copy number of the first compartment and the second compartment, calculating a first ratio (X) of the Δ copy number of the second amplification product (e.g., a “mean control” assay) to the amount of the first amplification product (e.g., a “mean test” assay) of the cellular RNA sample (e.g., the first compartment) and the EV RNA sample (e.g., the second compartment), and then calculating X. 细胞RNA With X EV RNA The second ratio. In some implementations, fragmentation of RNA transcripts in a sample is detected based on the second ratio.

[0222] In some embodiments, the first amplification product contains the inverse complementary sequence of a first RNA sequence of at most partially fragmented RNA transcripts (e.g., a "test" assay containing RNA regions expressed in both cellular and EV RNA compartments), and the second amplification product contains the inverse complementary sequence of a fragmented portion of the first RNA sequence (e.g., a "control").

[0223] In some embodiments, RNA transcripts are considered to be expressed in multiple cells and multiple extracellular vesicles, based in part on RNA sequencing data from multiple cells and RNA sequencing data from multiple extracellular vesicles / exosomes. In some embodiments, the separation between the first and second RNA fragments in the second RNA sequence is reflected in the RNA sequencing data from the second compartment, with a reduced number of sequencing reads mapping to the nucleotides in the second RNA sequence that separate the first and second RNA fragments compared to the number of sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.

[0224] 5.8. Design primers for detecting fragmentation of RNA transcripts This disclosure describes a method for designing primers for detecting fragmented RNA transcripts in a sample. Designing PCR assays targeting fragmented transcripts found in a sample or specific compartment (e.g., EV) is challenging because many fragments are small (less than 30 bases) and cannot accommodate forward and reverse PCR primers. The method described herein allows for the design of primers that overcome these limitations.

[0225] The method includes analyzing RNA sequencing data from RNA collected in a first compartment and RNA sequencing data from RNA collected in a second compartment. The RNA sequencing data from the first compartment contains a first RNA sequence that is at most partially fragmented, and the RNA sequencing data from the second compartment contains a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment. The first RNA sequence and the second RNA sequence are the same RNA transcript but are present in both the first and second compartments. The method also includes selecting a first primer pair capable of hybridizing with and amplifying the first RNA sequence, and a second primer pair capable of hybridizing with the first RNA fragment.

[0226] This disclosure describes a method for designing primers for detecting fragmentation of RNA transcripts. The method includes analyzing RNA sequencing data from RNA collected in a first compartment and RNA sequencing data from RNA collected in a second compartment. The RNA sequencing data from the first compartment contains a first RNA sequence that is at most partially fragmented; the RNA sequencing data from the second compartment contains a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment; and the first RNA sequence and the second RNA sequence are the same RNA transcript, but are present in both the first and second compartments. The method includes selecting a first primer pair capable of hybridizing with and amplifying the first RNA sequence; a second primer pair capable of hybridizing with and amplifying the first RNA fragment; and a third primer pair capable of hybridizing with and amplifying the second RNA fragment.

[0227] In some implementations, the first compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0228] In some implementations, the second compartment contains RNA from multiple cells, multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

[0229] In some implementations, the first compartment, the second compartment, or both contain RNA from a control sample.

[0230] In some implementations, the first compartment, the second compartment, or both contain RNA from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples.

[0231] In some implementations, environmental samples include water, wastewater, surface swabs, and air samples.

[0232] 5.9. Reagent Kit This disclosure describes a kit for performing a method for detecting RNA transcript fragmentation in a sample. In one embodiment, the kit includes a primer set comprising: a first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; and a second primer pair capable of hybridizing with and amplifying a first RNA fragment of a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0233] In another embodiment, the kit includes a primer set comprising: a first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most partially; a second primer pair capable of hybridizing with and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair capable of hybridizing with and amplifying a second RNA fragment of a second RNA sequence, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0234] In another embodiment, the kit includes a primer set, wherein the primer pairs are selected from: a first primer pair comprising a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11; ... The sequence comprises: a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 15; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 12; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 15 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 16; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 19; a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having at least 80% sequence identity with the sequence of SEQ ID NO: The reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; the forward primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and the reverse primer of the sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10;A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21; and instructions for performing any of the methods described herein.

[0235] 6. Example 6.1. Example 1. Transcript fragmentation assessment PCR assay In this work, RNA isolation and RNA-Seq library preparation reagents were used to analyze EV RNA, assessing the entire transcriptome from small miRNAs to large mRNAs and non-coding RNAs. This disclosure provides findings demonstrating significant differences between the EV transcriptome and the cellular transcriptome, with key differences in the transcript structure of many non-coding small RNAs. In this embodiment, data from a “transcript fragmentation assessment PCR assay” are provided, which can use qPCR or ddPCR to distinguish EV RNA from cellular RNA based on transcript structural differences. Such assays can quantify EV-specific transcripts, which can ultimately be used as biomarkers for monitoring human health.

[0236] 6.1.1. Method 6.1.1.1 Part 1: RNA Isolation 6.1.1.1.1 Cell Culture NTera-2 clone D1 was obtained from Millipore Sigma (Cat# 01071221-1VL). ATCC provided Jurkat, clone E6-1 (Cat# TIB-152), MCF7 (Cat# HTB-22), HEK293 (Cat# CRL-1573), and A549 cells (Cat# CCL-185). Cells were grown under ATCC-recommended culture conditions. Cells grown in suspension or adherent cultures were suitable for EV analysis by RNA-Seq. For cells grown in suspension culture, 1.4 mL of 70%–90% confluence cells was required for EV purification. For adherent cells, one well in a 6-well tissue culture plate was required for EV purification.

[0237] Cells are initially cultured under growth conditions recommended by the cell supplier or as previously determined. For example, one to three days before harvest, the cell culture medium is replaced with exosome-free FBS (System Biosciences Innovation, Cat# EXO-FBSHI-50A-1). At harvest, the cells are healthy and approximately 90% confluence. One to three days before EV harvest, the cell culture medium is replaced with exosome-depleted FBS (SBI). The cell density should be such that it reaches 70%–90% confluence at harvest.

[0238] 6.1.1.1.2 Precipitation of EVs from suspended cells For suspension cells grown for 1–3 days in FBS containing depleted exosomes, with 70%–90% confluence at EV harvest, the following steps are performed to precipitate the EVs: Collect 1.4 mL of suspended cells in a microcentrifuge tube.

[0239] As a negative control, 1.4 mL of culture medium containing exosome-depleted FBS (cell-free) was collected in a microcentrifuge tube.

[0240] Centrifuge the sample at 3,000 xg for 5 minutes in a microcentrifuge.

[0241] Transfer 1.2 mL of the supernatant to a new microcentrifuge tube, being careful not to transfer the precipitated cells.

[0242] As an optional step, the tube containing the precipitated cells can be preserved for harvesting cellular RNA as described in Section 3a.

[0243] Centrifuge 1.2 mL of sample at 12 k RPM for 15 minutes.

[0244] Transfer 1.0 mL of the supernatant to a new microcentrifuge tube, being careful not to transfer the precipitate. Store the sample on ice.

[0245] Add 200 mL of ExoQuick TC tissue culture medium exosome precipitation solution (SBI) to each sample. (Note: ExoQuick-TC is viscous and difficult to pipette. When using this reagent, use a wide-bore pipette tip for slow pipetting. Ensure that the full 200 mL volume is added to each sample.)

[0246] Vortex sample well. Visually inspect the samples to ensure they are properly mixed.

[0247] Store the sample at 4°C for at least 18 hours to allow EV to precipitate.

[0248] The precipitated EV samples can be stored at 4°C for up to 2 weeks.

[0249] Proceed to the "Isolation of EV RNA" section to isolate EV RNA.

[0250] 6.1.1.1.3 Precipitation of EVs from adherent cells For adherent cells grown for 1–3 days in FBS containing depleted exosomes, with 70%–90% confluence at EV harvest, the following steps are performed to precipitate EVs from the adherent cells: Cells grown in 6-well tissue culture plates are ideal for EV harvesting.

[0251] Collect 1.4 mL of conditioned cell culture medium in a microcentrifuge tube. Do not allow the pipette tip to touch the bottom of the plate.

[0252] As an optional step, plates containing adherent cells are preserved for harvesting cellular RNA as described in Section 3b.

[0253] As a negative control, 1.4 mL of culture medium containing exosome-depleted FBS (cell-free) was collected in a microcentrifuge tube.

[0254] Centrifuge the sample at 3,000 xg for 5 minutes in a microcentrifuge.

[0255] Transfer 1.2 mL of the supernatant to a new microcentrifuge tube, being careful not to transfer the precipitated cells.

[0256] Centrifuge 1.2 mL of sample at 12 k RPM for 15 minutes.

[0257] Transfer 1.0 mL of the supernatant to a new microcentrifuge tube, being careful not to transfer the precipitate. Store the sample on ice.

[0258] Add 200 μL of ExoQuick TC tissue culture medium exosome precipitation solution (SBI) to each sample. (Note: ExoQuick-TC is viscous and difficult to pipette. When using this reagent, use a wide-bore pipette tip to slowly pipette. Ensure that the entire 200 μL volume is added to each sample.)

[0259] Vortex sample well. Visually inspect the samples to ensure they are properly mixed.

[0260] Store the sample at 4°C for at least 18 hours to allow EV to precipitate.

[0261] The precipitated EV samples can be stored at 4°C for up to 2 weeks.

[0262] Proceed to the "Isolation of EV RNA" section.

[0263] 6.1.1.1.4 Isolation of cellular RNA from suspended cells Cellular RNA was isolated using the SingleShot Cell Lysis Kit (Bio-Rad Laboratories, Cat # 1725080 or 1725081). For RNA isolation from cells, the standard SingleShot protocol was used. 100 mL of the SingleShot cell lysis master mixture was required for each sample. The steps for isolating cellular RNA from suspension cells using the SingleShot Cell Lysis Kit are as follows: Prepare the SingleShot cell lysis master mixture by adding 10% of the sample amount as appropriate (Table 5). Store on ice.

[0264] Table 1. Preparation of the SingleShot cell lysis master mixture:

[0265] The starting material is the residual cells from step 6 of section 2a.

[0266] Add 1 mL of PBS to each residual cell sample. Vortex well.

[0267] Transfer 100 mL of cell sample into a new microcentrifuge tube.

[0268] Centrifuge the sample at 3,000 xg for 5 minutes in a microcentrifuge. Discard the supernatant.

[0269] The sample is pulsed and rotated to remove all trace amounts of residual liquid.

[0270] Add 100 mL of SingleShot cell lysis master mixture to each sample. Resuspend the precipitated EVs by vortexing.

[0271] Transfer the sample to a 0.2 mL PCR strip. Store on ice.

[0272] According to the scheme listed in Table 2, the samples were incubated in a PTC Tempo deep-hole thermal circulator (or equivalent device).

[0273] Table 2. Thermal circulation scheme. Set the heating cover temperature to 105℃.

[0274] Store EV RNA samples at -80°C.

[0275] The section on "Fragmentation of Cellular RNA" will be used to fragment cellular RNA.

[0276] 6.1.1.1.5 Isolation of cellular RNA from adherent cells Cellular RNA was isolated using the SingleShot Cell Lysis Kit (Bio-Rad Laboratories, Cat# 1725080 or 1725081). For RNA isolation from cells, the standard SingleShot protocol was used. 200 mL of the SingleShot cell lysis master mixture was required for each sample. The steps for isolating cellular RNA from adherent cells using the SingleShot Cell Lysis Kit are as follows: Prepare the SingleShot cell lysis master mixture by adding 10% of the sample amount as appropriate (Table 3). Store on ice.

[0277] Table 3. Preparation of Single-Shot Cell Lysis Master Mixture for Harvesting Cell RNA from Adherent Cells:

[0278] The starting material was adherent cells retained in the wells of a 6-well tissue culture plate from step 3 of the section “Precipitation of EVs from Adherent Cells”.

[0279] Add 1 mL of PBS to each well containing the cells to be analyzed. Mix gently. Aspirate the liquid completely.

[0280] Wash the cells again with 1 mL of PBS. Aspirate the liquid completely.

[0281] Add 200 mL of SingleShot cell lysis master mixture to each well. Gently tilt and rotate the plate to distribute the SingleShot master mixture evenly in the wells. Incubate at room temperature for 5 minutes.

[0282] Transfer the SingleShot cell lysis mixture to a 0.2 mL PCR strip. Store on ice.

[0283] The samples were incubated in a PTC Tempo deep-well thermal cycler (or equivalent device) according to the protocol listed in Table 2 of the “Isolation of cellular RNA from suspended cells” section.

[0284] Store EV RNA samples at -80°C.

[0285] Proceed to Section 5b to fragment cellular RNA.

[0286] 6.1.1.1.6 Isolation of EV RNA RNA samples from EVs were isolated for RNA-Seq using the SingleShot Cell Lysis Kit (Bio-Rad). 100 μL of SingleShot cell lysis master mixture was required for each sample. To isolate RNA from EVs, the RNA sample from the EVs was diluted 10-fold in SingleShot buffer containing DNase and proteinase K. The EV pellet was resuspended in 100 μL of SingleShot buffer containing DNase and proteinase K. The EVs were then incubated at 37°C for 5 minutes, followed by incubation at 75°C for 5 minutes. The samples were then cooled to 4°C and stored at -80°C.

[0287] Prepare a SingleShot cell lysis master mixture according to the appropriate amount of sample, as shown in Table 1, “Isolation of cellular RNA from suspension cells”. Store on ice.

[0288] Centrifuge the precipitated EV sample at 12 kJ / min for 15 minutes. Discard the supernatant.

[0289] The sample is pulsed and rotated to remove all trace amounts of residual liquid.

[0290] Add 100 µL of SingleShot cell lysis master mix to each sample. Resuspend the precipitated EVs by vortexing. Store on ice.

[0291] Incubate the samples according to the instructions in Table 6 of Section 3a.

[0292] Store EV RNA samples at -80°C.

[0293] Proceed to the "Preparation of EV RNA" section 6.1.1.2 Part 2: Preparation of RNA-Seq Libraries 6.1.1.2.1 Preparation of EV RNA This section describes the preparation of RNA-Seq libraries for EV RNA. The steps are as follows: Before use, completely thaw the SEQuoia Complete reagent AE and RNA sample. Store on ice.

[0294] Prepare the RNA fragmentation master mixture by adding 10% of the sample to an appropriate amount (Table 4). Store on ice.

[0295] Table 4. Preparation of the RNA fragmentation master mixture

[0296] In a 0.2 mL PCR tube, aliquot 13 μL of the RNA fragmentation master mixture. Store on ice.

[0297] Add 5 μL of EV RNA sample to each tube.

[0298] Gently vortex the mixed test tube, then centrifuge to collect the reaction at the bottom of the tube.

[0299] Store the tubes on ice.

[0300] Proceed to the "End-of-Line Repair" section.

[0301] Note that EV RNA samples are not fragmented prior to RNA-Seq library preparation, while cellular RNA is fragmented. If analyzing both EV and cellular RNA samples, ensure they are in separate 0.2 mL PCR strips.

[0302] 6.1.1.2.2 Fragmentation of cellular RNA This section describes the preparation of RNA-Seq libraries for cellular RNA. The steps are as follows: Before use, completely thaw the SEQuoia Complete reagent AE and RNA sample.

[0303] Preheat the thermal circulator to 94°C and the heating cover to 105°C.

[0304] Prepare the RNA fragmentation master mixture according to the instructions in Table 4 of the “Preparation of EV RNA” section.

[0305] In a 0.2 mL PCR tube, aliquot 13 μL of the RNA fragmentation master mixture. Store on ice.

[0306] Add 5 μL of cellular RNA sample to each tube.

[0307] Gently vortex the mixed test tube, then centrifuge to collect the reaction at the bottom of the tube.

[0308] According to the scheme in Table 5, incubate the tubes in a PTC Tempo deep-hole thermal circulator (or equivalent device).

[0309] Table 5. Thermal circulation scheme. Set the heating cover temperature to 105℃.

[0310] Store the tubes on ice.

[0311] Proceed to the "End-of-Line Repair" section.

[0312] 6.1.1.2.3 End-of-trim The "end-of-pipe remediation" solution is provided in the following steps: Preheat the thermal circulator to 37°C and the heating cover to 105°C.

[0313] Add 2 μL of reagent B and terminal repair enzyme to each sample.

[0314] The reactants are mixed by vortexing. The sample is then collected at the bottom of the tube by pulse centrifugation.

[0315] According to the scheme in Table 6, incubate the tubes in a PTC Tempo deep-hole thermal circulator (or equivalent device).

[0316] Table 6. Thermal circulation scheme. Set the heating cover temperature to 105℃.

[0317] Place the reaction on ice for 5 minutes.

[0318] Proceed to the "Poly(A) with tail" section.

[0319] 6.1.1.2.4 Poly(A) with tailing The solution for Poly(A) tailing is provided in the following steps: Preheat the thermal circulator to 16°C and the heating cover to 105°C.

[0320] Prepare a Poly(A) tailings master mixture by adding 10% of the sample amount as appropriate (Table 7). Store on ice.

[0321] Table 7. Preparation of Poly(A) tailings-based mixtures

[0322] Add 25 μL of poly(A) master mixture to each reaction.

[0323] The reactants are mixed by vortexing. The sample is then collected at the bottom of the tube by pulse centrifugation.

[0324] According to the scheme in Table 8, incubate in a PTC Tempo deep-hole thermal circulator (or equivalent device).

[0325] Table 8. Thermal circulation scheme. Set the heating cover temperature to 105℃.

[0326] Place the reaction on ice for 5 minutes.

[0327] Proceed to Chapter 8, and continue the synthesis.

[0328] 6.1.1.2.5 Continuous Synthesis Preheat the thermal circulator to 34°C and the heating cover to 105°C.

[0329] Add 5 μL of reagent E, SEQzyme mixture to each reaction.

[0330] The reactants are mixed by vortexing. The sample is then collected at the bottom of the tube by pulse centrifugation.

[0331] According to the scheme in Table 9, incubate in a PTC Tempo deep-hole thermal circulator (or equivalent device).

[0332] Table 9. Thermal circulation scheme. Set the heating cover temperature to 105℃.

[0333] Place the reaction on ice for 5 minutes.

[0334] This is the safe stop point. Samples can be stored overnight at 4°C, or if stored for more than 24 hours, at -20°C.

[0335] When ready, proceed to the "cDNA Purification" section.

[0336] 6.1.1.2.6 cDNA Purification Allow the purification beads to reach room temperature.

[0337] The vortex beads are brought back to levitate.

[0338] Add 20 μL of purification beads to each sample.

[0339] The mixing hole is moved up and down several times.

[0340] Incubate at room temperature for 20 minutes.

[0341] Place the tube on the magnetic rack for 5-10 minutes or until the solution becomes clear.

[0342] Transfer the supernatant containing cDNA to a new 0.2 mL PCR tube.

[0343] Proceed to the "cDNA Concentration" section.

[0344] 6.1.1.2.7 cDNA Concentration 1. Allow the SPRIselect reagent (Beckman Coulter) to reach room temperature.

[0345] 2. At the end of the cDNA purification step (Section 9, Step 7), add 112 μL of SPRIselect reagent to the collected supernatant.

[0346] 3. Mix the holes several times by moving them up and down.

[0347] 4. Incubate at room temperature for 5-8 minutes.

[0348] 5. Briefly centrifuge the reaction to collect the reactants at the bottom of the tube.

[0349] 6. Place the tube on the magnetic rack for 5 minutes or until the solution becomes clear.

[0350] Note: Until the step “Let the beads dry at room temperature for 2-4 minutes. Do not over-dry the beads”, keep the tube on the magnetic rack.

[0351] 7. Aspirate and discard the clear solution.

[0352] 8. Wash the beads by adding 200 μL of freshly prepared 80% ethanol. Incubate for 30 seconds.

[0353] 9. Aspirate and discard the ethanol without affecting the beads.

[0354] 10. Repeat the ethanol washing steps (steps 8 and 9) to remove all trace amounts of ethanol.

[0355] 11. Allow the beads to dry at room temperature for 2-4 minutes. Do not over-dry the beads.

[0356] 12. Remove the tube from the magnetic rack.

[0357] 13. Add 24 μL of IDTE, pH 8.0 to each tube and mix by moving the tube up and down several times through the mixing hole.

[0358] 14. Incubate the tube at room temperature for 2 minutes.

[0359] 15. Briefly centrifuge the reaction mixture, placing the tube on a magnetic rack for 5 minutes or until the solution becomes clear.

[0360] 16. Transfer 22.5 μL of supernatant containing cDNA to a new 0.2 mL PCR tube.

[0361] This is the safe stop point. Samples can be stored overnight at 4°C, or if stored for more than 24 hours, at -20°C.

[0362] When ready, proceed to the section on "Determining the Number of PCR Cycles for Library Amplification".

[0363] 6.1.1.2.8 Determination of the number of qPCR cycles for library amplification For samples treated with qPCR, the inventors of this application discovered that EV RNA is significantly different from cellular RNA and a real-time PCR step is needed to determine the optimal number of PCR cycles for library amplification. Cellular RNA samples also benefit from this step. The steps are as follows: SYBR green I nucleic acid gel dye (ThermoFisher) is in high concentration in DMSO. Thaw at room temperature and dilute to 2X in DMSO.

[0364] Thaw RNA samples and cell-free control samples on ice. Thaw amplification mixture (green cap) from the SEQuoia Complete kit and individual SEQuoia double-index primers, one per sample, on ice.

[0365] Prepare the library PCR master mixture by adding 10% of the sample amount as appropriate (Table 10). Store on ice.

[0366] Table 10. Preparation of the master mixture for library PCR

[0367] Distribute 36.5 μL of the library PCR master mixture equally into each well of a 96-well PCR plate, including wells containing cell-free control samples.

[0368] Add 11 μL of the sample from the “cDNA Concentration” section, and follow the step “Transfer 22.5 μL of supernatant containing cDNA to a new 0.2 mL PCR tube”.

[0369] Carefully puncture the wells of the 96-well dual-index primer plate and add 2.5 μL of SEQuoia dual-index primer to each well of the PCR plate. Ensure that each well yields a different index primer.

[0370] Seal the plate with an optical sealing film. Vortex the plate and centrifuge at 4000 rpm for 2 minutes to collect the reactants at the bottom of the plate and eliminate air bubbles.

[0371] This experiment should be performed using a CFX Opus 96 real-time PCR instrument (Bio-Rad) running CFX Maestro software version 2.0 (or later). Other real-time PCR instruments may also be used.

[0372] Run the RT-PCR reaction plate on a Bio-Rad CFX Opus 96 or equivalent real-time PCR instrument according to the thermal cycling protocol listed in Table 11. Ensure signal detection is limited in the SYBR channel.

[0373] Table 11. Thermal Cycling Scheme

[0374] To determine the number of library amplification cycles, two different thresholds were used to analyze the data. These thresholds effectively set the upper and lower boundaries of the cycles during which exponential amplification occurred. This is described in the following sections: After running RT-PCR, open the executable file using CFX Maestro software.

[0375] A rough estimate of the maximum RFU of the amplification trace. For Figure 2B The exemplary amplification curves shown use 30,000 RFU as the maximum value (red, horizontal line), even though some amplification traces have a maximum value higher or lower than 30,000 RFU.

[0376] Set the baseline threshold to 25% of the maximum RFU and collect Cq values ​​in integer form.

[0377] Next, collect the Cq value in the CFX Maestro program.

[0378] Set the baseline threshold to 75% of the maximum RFU and collect Cq values ​​in integer form. Figure 3A The amplification curves (purple, horizontal lines) are shown with baseline thresholds set to 25% and 75% of the maximum RFU. Table 12 shows the integer Cq values ​​for each sample at 25% and 75% of the maximum RFU.

[0379] Table 12. Cq values ​​at 25% and 75% of the maximum RFU.

[0380] The number of PCR cycles to be performed for library amplification should be between the Cq values ​​(inclusive) at 25% and 75% of the maximum RFU. The number of amplification cycles can vary between samples. For simplicity, samples are grouped to minimize the number of library amplification runs. In the examples shown in Table 13, three library amplification experiments should be performed using 10, 13, and 16 amplification cycles. Samples 9 and 11 can be run with 10 or 13 amplification cycles. Sample 10 can be run with 13 or 16 amplification cycles.

[0381] Table 13. Number of library amplification cycles performed for each sample

[0382] Proceed to the "RNA-Seq Library Amplification and Indexing" section.

[0383] 6.1.1.2.9 RNA-Seq Library Amplification and Indexing 1. Use the data collected in the “Determination of the Number of Library Amplification PCR Cycles” section to determine how many individual library amplification PCR runs are required. For the example experiments shown in Table 13, three library amplification PCR runs (10, 13, and 16 cycles) are required.

[0384] 2. Collect an appropriate RNA sample for a single library amplification PCR run and thaw it on ice.

[0385] 3. Thaw the amplification mixture (green cap) from the SEQuoia Complete kit, along with the same SEQuoia double-index primers used in the sample in Section 11. Store on ice.

[0386] 4. Prepare the RNA-Seq library amplification master mixture by adding 10% of the sample as needed (Table 14). Store on ice.

[0387] Table 14. Preparation of the master mixture for RNA-Seq library amplification

[0388] 5. Divide the 36.5 μL library PCR master mixture into 0.2 mL strip tubes.

[0389] 6. Add the 11 μL sample from step 16 of section 10 to each tube.

[0390] 7. Add 2.5 μL of SEQuoia double-index primer to each tube. Ensure that each sample has the same double-index primer as described in the section on "Determination of the Number of PCR Cycles for Library Amplification".

[0391] 8. Amplify the samples on a PTC Tempo deep-hole thermal cycler (or equivalent) according to the protocol listed in Table 15.

[0392] Table 15. RNA-Seq library amplification protocols

[0393] Repeat the library amplification PCR run with different numbers of PCR cycles (if needed), steps 2-8.

[0394] Proceed to the "Post-Amplification Cleanup" section.

[0395] 6.1.1.2.10 Post-amplification cleanup 1. Allow the SPRIselect reagent (Beckman Coulter) to reach room temperature.

[0396] 2. Add 60 μL of SPRIselect reagent to each tube.

[0397] 3. Mix the holes several times by moving them up and down.

[0398] 4. Incubate at room temperature for 5-8 minutes.

[0399] 5. Briefly centrifuge the reactants to collect them at the bottom of the tube.

[0400] 6. Place the tube on the magnetic rack for 5 minutes or until the solution becomes clear.

[0401] Note: Keep the tube on the magnetic rack until step 11.

[0402] 7. Aspirate and discard the clear solution.

[0403] 8. Wash the beads by adding 200 μL of freshly prepared 80% ethanol. Incubate for 30 seconds.

[0404] 9. Aspirate and discard the ethanol without affecting the beads.

[0405] 10. Repeat the ethanol washing steps (steps 8 and 9) to remove all trace amounts of ethanol.

[0406] 11. Allow the beads to dry at room temperature for 2-4 minutes. Do not over-dry the beads.

[0407] 12. Remove the tube from the magnetic rack.

[0408] 13. Add 24 μL of IDTE, pH 8.0 to each tube and mix by moving the tube up and down several times through the mixing hole.

[0409] 14. Incubate the tube at room temperature for 2 minutes.

[0410] 15. Briefly centrifuge the reaction mixture, placing the tube on a magnetic rack for 5 minutes or until the solution becomes clear.

[0411] 16. Transfer 18 μL of supernatant containing the RNA-Seq library to a new PCR tube.

[0412] 17. This is the safe stop point. Samples can be stored overnight at 4°C, or if stored for more than 24 hours, at -20°C.

[0413] 18. Conduct the section on "Evaluation of the quality and quantity of RNA-Seq libraries".

[0414] 6.1.1.2.11 Assessment of the quality and quantity of the library According to the manufacturer's instructions, the quality and quantity of RNA-Seq libraries are determined using the Agilent 2100 Bioanalyzer or an equivalent device with a high-sensitivity DNA chip. Exemplary bioanalyzer traces for analyzing cellular RNA-Seq libraries or EV RNA-Seq libraries are shown below. Figure 4 and 3B middle.

[0415] 6.1.1.3 Determination and Primers Table 16 lists the primers, and Table 17 lists the assays including primer combinations.

[0416] A "+" indicates that the following bases are locked nucleic acids (LNAs).

[0417] 6.1.1.4 Part 4: Processing RNA-Seq Data 6.1.1.4.1 Seq Sense Data Analytics Pipeline There are three steps to processing and analyzing SEQuoia complete strand RNA data: 1. FASTQ pretreatment: Pruning of SEQuoia intact and specific Poly(A) tails 2. Alignment: A single alignment is performed between a set of transcriptomes and the merged annotation set.

[0418] 3. Feature Counting – Mapped read output of raw and normalized counts (TPM, RPKM) Download the FASTQ file generated on the instrument or Illumina Basespace and analyze the RNA sequencing data using the web-based Seq-Sense platform (https: / / seqsense.bio-rad.com / ).

[0419] Launch the Seq-Sense platform and select the SEQuoia Complete kit as the target kit.

[0420] Upload and retrieve the FASTQ file from the instrument or Basespace server.

[0421] Pipeline parameters were then set and processed for experiments. In this study, hg38 was used as the species, and UMI processing was enabled to remove duplicates from reads (both R1 and R2 files were available). R1 ​​suggested a read length of 75 bp, and R2 suggested a read length of 8 bp. The first 8 bases of R2 consisted of a random tag sequence that could be used as a unique molecular identifier (UMI).

[0422] 6.1.2. Part 5: Analysis of RNA-Seq Data RNA-Seq FASTQ data were processed using SeqSense NGS data analysis software (Bio-Rad). RNA transcript structure was determined by converting the output BAM file into a bigWig file (interval size = 1) and visualizing the bigWig file as a custom trace on the UCSC Genome Browser.

[0423] 6.1.2.1 Target gene read analysis Upload the sequencing output BAM file to the Galaxy server (https: / / usegalaxy.org / ).

[0424] In the submenu, select bam as the file type and Human 2013 (hg38) as the target genome.

[0425] Next, the BAM reads were converted into the Bigwig format, which is easy to visualize on the UCSC Genome Browser.

[0426] Set the tool parameters to a range size of 1 (based on bases) and hg38 as the target genome.

[0427] The bigwig file is visualized on the UCSC Genome Browser, such as... Figure 1A As shown. Under the custom trace, the configuration is set to "full". The trace automatically scales to display gene expression levels.

[0428] Record the gene expression pattern of the target gene.

[0429] 6.1.2.2 Analysis of transcript structure 6.1.2.2.1 PCR assay design Based on RNA-Seq data, a PCR assay set for transcript fragmentation assessment was designed, targeting both intact transcripts in cellular RNA and fragmented transcripts in EV RNA. RNA transcript structure was determined by visualizing cellular RNA and EV RNA expression using the bigWig trace on the UCSC Genome Explorer. Each assay set consisted of two types of assays (control assay and test assay). The control assay amplified RNA regions expressed in both cellular and EV RNA. The test assay amplified RNA regions expressed in cellular RNA but less expressed in EV RNA (because it is fragmented). qPCR method: qPCR reaction steps -1L). Each PCR assay may have more than one control or test assay. If more than one control or test assay is used to evaluate the target, the average Cq value of the PCR reaction is taken. Six PCR assays were developed. The oligonucleotide sequences of each primer are listed in Table 16, and the assays are listed in Table 17.

[0430] 6.1.2.2.2 PCR analysis of cellular and EV RNA RNA isolated from cells and EVs used for PCR analysis was analyzed using the iTaq Universal SYBR Green One-Step Kit (Bio-Rad, Cat# 1725150 or 1725151) according to the manufacturer's instructions. To estimate the level of genomic DNA background, each sample was analyzed with and without reverse transcriptase. The Cq value of samples analyzed without reverse transcriptase reflects the level of background DNA. In all samples, the level of contaminating DNA was negligible and did not affect the PCR results (Table 3).

[0431] qPCR method: qPCR thermocycling When using qPCR, RNA structure is assessed using the ΔCq values ​​measured by the control and the test. Measurements of EV RNA are expected to have large negative ΔCq values, while measurements of cellular RNA are expected to have relatively small ΔCq values, which can be either negative or positive.

[0432] The qPCR steps using the iTaq Universal SYBR Green One-Step kit are as follows: Collect appropriate RNA samples for qPCR validation of RNA samples and thaw them on ice.

[0433] Thaw iTaq Univ SYBR Green 1-Step and iScript reverse transcriptase, and perform 4x assays. Store on ice.

[0434] Prepare the qPCR master mixture by adding 10% of the sample to the appropriate amount (Table 18). Store on ice.

[0435] Table 18. Preparation of the master mixture for qPCR validation

[0436] Distribute 20.25 μL of the library PCR master mixture equally into each well of a 96-well PCR plate, including wells containing cell-free control samples.

[0437] The plate was sealed with an optical sealing film. The plate was vortexed and centrifuged to collect the reactants at the bottom of the plate.

[0438] Figures 5A-5L When using qPCR, run RT-PCR reaction plates on a Bio-Rad CFX Opus 96 or equivalent real-time PCR instrument according to the thermal cycling protocol listed in Table 19.

[0439] Table 19. Thermal Cycling Scheme

[0440] After generating cDNA in the first 50°C step, the protocol immediately performs PCR amplification and monitors fluorescence. After the RT-PCR run, the run file is opened using CFX Maestro software, and Cq values ​​are collected from the "Quantitative Tag".

[0441] The assay of EV RNA is expected to have a large negative ΔCq value.

[0442] The assay of cellular RNA is expected to have a relatively small ΔCq value, which can be negative or positive.

[0443] 6.1.3. qPCR Results 6.1.3.1 EV transcripts are fragmented When comparing RNA-Seq results from RNA isolated from EV particles with those from cellular RNA analysis, significant differences were revealed. Many EV transcripts were observed to be fragmented compared to their intact cellular counterparts. Furthermore, the pattern of EV transcript fragmentation was generally consistent across different cell lines (see [link to original text]). Figures 5A-5L ).

[0444] Several patterns of EV transcript fragmentation were observed. Figure 5J One example of the pattern is composed of separate RNA fragments at the 5' and 3' ends of the transcript, with a few transcripts containing bases in the middle. Figure 5K and Figure 5LAnother illustrative pattern shows the EV transcript, which contains the middle portion of the gene and has a few bases at the 5' and 3' ends. The EVSCARNA5 transcript consists of two regions in the middle of the gene and a few bases at the 5' and 3' ends, and is a combination of the two transcript fragmentation patterns mentioned above (see [link to original text]). Figures 5A-5L ).

[0445] 6.1.3.2 PCR assay to distinguish EVs and cellular RNA based on transcript structure The fragmented transcriptomic structures of EV RNA observed via RNA-Seq are intriguing. To provide further evidence for the presence of these structures, novel types of assays were developed using PCR technology: “transcript fragmentation assessment PCR assays.” This assay strategy involved designing control PCR assays that amplified RNA regions present in both EV and cellular RNA, and test PCR assays that, based on RNA-Seq data, should amplify cellular RNA but not EV RNA (because the amplicons contain RNA regions largely absent in EVs due to transcriptional fragmentation). RNA transcript structure was determined by comparing the ΔCq values ​​of the control and test assays. Assays analyzing EV RNA were expected to have large ΔCq values ​​because control assays should amplify EV RNA and have Cq values, while test PCR assays should not amplify EV RNA well and have Cq values ​​significantly delayed compared to controls. When analyzing cellular RNA with these assays, relatively small ΔCq values ​​were expected because both control and test assays should amplify intact transcripts with similar Cq values. Therefore, these assay sets assessed transcript structures with large negative ΔCq values ​​reflecting fragmented transcripts and small ΔCq values ​​suggesting intact transcripts. The ΔΔCq value is used to quantify the differences in target gene transcription structure between cellular compartments and EV compartments by comparing EV and cellular ΔCq values.

[0446] Designing PCR assays targeting fragmented transcripts found in EVs can be challenging because many fragments are small (less than 30 bases) and cannot accommodate forward and reverse PCR primers. In some cases, we incorporate locked nucleic acid bases into PCR primers to increase primer Tm and improve PCR performance. Despite the difficulties in designing PCR primers, [the following is a separate, unrelated sentence:] ... Figures 6A-6D The six targets shown were used to design a PCR assay for transcript fragmentation assessment. The PCR assay design and results are shown in... Figures 7A-7D (RNY5) Figures 8A-8D (SCARNA10) Figures 9A-9D (SCARN5A) Figures 10A-10D (SCARNA10) Figures 11A-11D (RMRP) and Figures 6A-6D (RNY1).

[0447] Targeting RNY5 (Figures 7A-7D ), RNU12 ( Figures 8A-8D ), SCARNA5 ( Figures 9A-9D ), SCARNA10 ( Figures 10A-10D ) and RMRP ( Figure 6A The transcript fragmentation assessment PCR assay confirmed the fragmented transcript structures of these target genes in EVs observed by RNA-Seq (respectively, ) Figures 11A-11D , 7A (8A, 9A, and 10A). The ΔΔCq values ​​in different cell lines ranged from -5.40 to -14.69, indicating that the prevalence of fragmented transcript structures in EV RNA was at least 42 times higher than that in cellular RNA.

[0448] The RNY1 transcript exhibits distinct 5' and 3' fragments in the EVs of all analyzed cell lines. Figures 6A-6D However, the ΔΔCq values ​​range from -1.93 to -4.83, which makes the RNY1EV transcript structure closer to cellular RNA than other targets assessed by transcript fragmentation for PCR quantification (ΔΔCq ranges from -5.40 to -14.69; see RNY5). Figures 7A-7D ), RNU12 ( Figures 8A-8D ), SCARNA5 ( Figures 9A-9D ), SCARNA10 ( Figures 10A-10D ) and RMRP ( Figure 11A Visual examination of the RNY1 RNA-Seq read map indicated that the transcript was not fully fragmented, which could reflect a small negative ΔΔCq score (see [link to RNY1 RNA-Seq read map]). Advances in Experimental ).

[0449] 6.1.4. Discussion This data provides evidence that EV transcripts are fragmented. Previous work analyzing Y-RNA supports this theory. Y-RNA is a small non-coding RNA that interacts with RO60 and SSB proteins to form a ribonucleoprotein (RNP) complex, which is functionally involved in DNA replication, RNA quality control, and cellular stress responses (Valkov, N., Medicine and Biology RNASpringer, 2020, pp. 327-342. doi: 10.1007 / 978-981-15-1671-9_20). Four human Y-RNAs encoded by the RNY1, RNY3, RNY4, and RNY5 genes exist. The structure of the RNY5 transcript isolated from EVs of human BJ and K562 cells has been previously determined by Northern blot analysis and found to have the same split 5' and 3' structures observed in our RNA-Seq results (Chakrabortty, SK, Springer, 2020, pp. 327-342. doi: 10.1007 / 978-981-15-1671-9_20). Figures 12A-12B , 21(11):1966-1979 (2015))( Figures 12A-12B This provides further evidence from Northern blotting that the EV transcript is fragmented in human samples. Furthermore, the location of the transcript fragments coincides with the locations observed in our RNA-Seq experiments, providing additional support for the existence of fragmented RNY5 EV transcript structures (see [link to relevant documentation]). Cell ).

[0450] Prior work using X-ray crystallography to analyze y-RNA / RO60 interactions provided insights into how transcript fragmentation occurs in some cases, which helps interpret the data in the current study. Mapping y-RNA bases crucial for RO60 protein interactions to the 8-base sequence at the 5' end and the 7-base sequence at the 3' end of the y-RNA transcript (Stein, AJ) Figure 13A , 121(4): 529–539, May 2005) ( Figures 13B-13E The 5' sequence motif is conserved in all RNY genes and begins at the fourth base from the 5' end of the transcript. The first six bases of the RO60 3' binding site are also conserved in all RNY genes. However, the final base of the 3' motif is not conserved in the human RNY4 gene; it is G instead of A. Figure 5E Interestingly, all RO60 interaction sites were found in the transcript regions highly expressed in EVs. This suggests the possibility that RO60 binds to RNY transcripts to protect the binding region from nuclease digestion. RMRP is abundant in EVs (see [link to EV]). Hum Mol Genet , 10A -10D) is the RNA component of the RNP complex with ribonuclease activity (Hermanns, P., Physiol Genomics , 14(23): 3723-3740 (2005)). It appears that the ribonuclease activity in EVs can digest unprotected RNA and fragmented RNY transcripts into fragments observed by RNA-Seq.

[0451] Perhaps importantly, all the fragmented EV transcripts identified in this study were small non-coding RNAs, which are components of the functional RNP complex. The fragmented transcript fragments found in EVs may be protected from digestion by ribonucleases due to protein binding. Distinguishing between free-floating RNA and RNA that is a component of the functional RNP complex is an important factor to consider when understanding the EV transcriptome.

[0452] The RNY gene has been found to be enriched in human blood (Dhahbi, JM, Cancers , 45: 990–998 (2013), doi: 10.1152 / physiolgenomics.00129.2013), and is investigating its use as a biomarker for monitoring human health (C. Gulìa et al., “ J Extracell Vesicles , 12(5) (2020) doi: 10.3390 / cancers12051238.) and Driedonks et al., Figure 1C , 9(1) (2020), doi:10.1080 / 20013078.2020.1764213). Transcript fragmentation assessment PCR assays for RNY1 and RNY5 may be a useful tool for tracking these markers.

[0453] 6.2. Example 2: Transcript Fragmentation Assessment by ddPCR Assay This experiment evaluates the primer design and test / control assays used in Example 1 in droplet digital PCR assays based on transcript structural differences to analyze and differentiate EV RNA and cellular RNA.

[0454] like Figures 14-19 As shown, EV RNA and cellular RNA samples were cultured, precipitated, separated, and lysed as described in Example 1. After lysing the EV RNA and cellular RNA samples, cDNA was prepared from the EV and cell SingleShot lysates.

[0455] 6.2.1.1 Preparation of cDNA The iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad #1725035) was used to prepare cDNA in large quantities from EV lysates (14 μl of undiluted lysate added to the reaction) and cell lysates (1:50 diluted in TE). It is a two-step method: cDNA synthesis is performed after gDNA cleanup.

[0456] The steps for preparing cDNA are provided below: Preparation of DNase master mixture: Table 20: DNA Master Mixture

[0457] Add 2 μl of the mixture to 14 μl of the sample (if less than 14 μl, bring the total to 14 w / H2O). After mixing, place it in a thermal cycler: Table 21: DNase Reaction Scheme in Thermal Cyclist

[0458] Add 4 μl of RT Supermix to each 16 μl reaction mixture and place in a thermal cycler: Table 22: cDNA Synthesis Reaction Scheme in Thermal Cyclist

[0459] Transfer the supernatant containing cDNA into a new PCR tube.

[0460] 6.2.1.2 ddPCR reaction setup After cDNA synthesis, a ddPCR reaction was established using the ddPCR Evagreen Supermix. EV cDNA was directly used in a 20 μl ddPCR reaction (QX200 ddPCR EvaGreen Supermix, Bio-rad #186-4034) containing 250 nM primers (5 μl). The Evagreen Supermix was used according to the manufacturer's instructions. The QX200 ddPCR EvaGreen Supermix is ​​a 2x concentrated, ready-to-use reaction mixture containing all components required for droplet digital PCR except for primers and template. 250 nM primers were added to the reaction.

[0461] Dilute the cell cDNA 1:2 in water, take 5 μl for 20 μl EvaGreen reaction, and add 250 nM primer.

[0462] The preparation of the reaction mixture is provided as follows: Table 23: Preparation of the reaction mixture:

[0463] Table 24: Preparation of the main mixture:

[0464] 6.2.1.3 Droplet formation Droplets were generated using an automated droplet generator (1864101) with QX200 Droplet Generation Oil for EvaGreen (Bio-Rad #1864006). Approximately 20,000 drops were produced from a 20 μL reaction. The droplet generator used a specific oil to form droplets. Once the droplets were formed, they were placed on a 96-well plate.

[0465] Take the average of the droplet counts from two identical wells. If more than one control or test is used, take the average of the droplet counts.

[0466] 6.2.1.4 Thermal Cycling Thermal cycling was performed in a PTC Tempo deep-well thermal cycler (Bio-Rad #12015392) according to the reagent instructions and manufacturer's instructions. This amplified cDNA from EV RNA and cellular RNA samples while simultaneously hardening and stabilizing the droplets.

[0467] 6.2.1.5 Droplet Reading Count the droplets on the QX200 Droplet Digital PCR System (or any Bio-Rad droplet reader, including the QX600). Analyze the data using QX Manager software.

[0468] Use the reading oil to read the droplet. Pass all contents of the orifice through the reader.

[0469] When both positive and negative droplets are present, the QX software determines a threshold. For example, if only positive droplets are present, the cellular RNA template needs to be further diluted and repeated.

[0470] 6.2.1.6 Data Analysis As described in Example 1, “Design of PCR Assays,” transcript fragmentation assessment PCR assays were designed based on RNA-Seq data, targeting intact transcripts in cellular RNA and fragmented transcripts in EV RNA. RNA transcript structure was determined by visualizing cellular RNA and EV RNA expression using the bigWig trace on the UCSC Genome Browser. Each assay set consisted of two assay types (control assay and test assay). The control assay amplified RNA regions expressed in both cellular and EV RNA compartments. The test assay amplified RNA regions expressed in cellular RNA but less expressed in EV RNA (because it is fragmented). Each PCR assay set may have more than one control or test assay.

[0471] like Figure 14As shown, transcript fragmentation using ddPCR is determined through the following steps: First, the “Δcopy number” is calculated, which is the amount of the second amplified product of the first fragment of the second RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “average control” – “average test”). Then, the cellular RNA sample (e.g., first compartment X) is calculated. (细胞RNA) ) and EVRNA samples (e.g., second compartment X) (EV RNA) The first ratio (X) of the "Δ copy number" and the "average test" of X, and X (细胞RNA) With X (EV RNA) The second ratio. The first amplification product contains the inverse complementary sequence of the first RNA sequence of at most partially fragmented RNA transcript (e.g., a "test" assay containing the RNA region expressed in the cell and EV RNA compartment), and the second amplification product contains the inverse complementary sequence of the fragmented portion of the first RNA sequence (e.g., a "control").

[0472] For example, used in ddPCR Figure 15 The ratio of the second to the first amplification product in the A549 cell line shown by RMRP indicates that the RMRP transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to the qPCR results.

[0473] For example, used in ddPCR Figure 16 The ratio of the second amplification product to the first amplification product in the RNY5, A549 cell line shown indicates that the RNY5 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0474] For example, used in ddPCR Figure 17 The ratio of the second amplification product to the first amplification product in the RNU12, A549 cell line shown indicates that the RNU12 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to the qPCR results.

[0475] For example, used in ddPCR Figure 18 The ratio of the second to the first amplified product in the SCARNA5 A549 cell line shown indicates that the SCARNA5 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0476] For example, used in ddPCR Figure 19 The ratio of the second to the first amplified product of SCARNA10 in the A549 cell line shown indicates that the SCARNA10 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to the qPCR results.

[0477] For example, used in ddPCR ​ The ratio of the second amplification product to the first amplification product in the RNY1, A549 cell line shown indicates that the RNY1 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to the qPCR results.

[0478] Equivalent and incorporated by reference Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A method for detecting fragmentation of RNA transcripts in a sample, the method comprising: Add a first primer pair and a second primer pair to the sample, wherein The sample contains: The first RNA sequence of the RNA transcript, which is at most partially fragmented; The second RNA sequence of the RNA transcript, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and an optional second RNA fragment. The first primer pair is capable of hybridizing with the first RNA sequence and amplifying all or part of the first RNA sequence; The second primer pair can hybridize with the first RNA fragment and amplify the first RNA fragment; The first RNA sequence and the first RNA fragment are amplified using the first and second primer pairs to generate a first amplification product containing the first RNA sequence or its inverse complementary sequence and a second amplification product containing the first RNA fragment or its inverse complementary sequence. Quantify the amount of the first amplification product and the amount of the second amplification product; as well as The fragmentation of the RNA transcript in the sample is detected based on the amount of the second amplification product, wherein the amount of the second amplification product indicates RNA transcript fragmentation.

2. The method according to claim 1, wherein the second RNA sequence is a fragment of the first RNA sequence.

3. The method of claim 1, wherein the detection comprises determining the amount of fragmentation of the RNA transcript in the sample based on the amount of the second amplification product, wherein a comparison between the amount of the second amplification product and the amount of the first amplification product indicates the amount or extent of RNA transcript fragmentation.

4. A method for detecting fragmentation of RNA transcripts in a sample, the method comprising: At least a first primer pair, a second primer pair, and a third primer pair are added to the sample, wherein... The sample contains: The first RNA sequence of the RNA transcript, which is at most partially fragmented; and The second RNA sequence of the RNA transcript, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and an optional second RNA fragment. The first primer pair is capable of hybridizing with the first RNA sequence and amplifying all or part of the first RNA sequence; The second primer pair is capable of hybridizing with and amplifying the first RNA fragment; and The third primer pair is capable of hybridizing with the second RNA fragment and amplifying the second RNA fragment; The first RNA sequence, the first RNA fragment, and the second RNA fragment are amplified using the first, second, and third primer pairs, respectively, to generate a first amplification product containing the first RNA sequence or its reverse complementary sequence, a second amplification product containing the first RNA fragment or its reverse complementary sequence, and a third amplification product containing the second RNA fragment or its reverse complementary sequence. Quantitative: The amount of the first amplified product, The amount of the second amplification product; and The amount of the third amplification product; as well as The fragmentation of the RNA transcript in the sample is detected based on the average amount of the second amplification product and the third amplification product, wherein the average amount of the second amplification product and the third amplification product indicates RNA transcript fragmentation.

5. The method of claim 4, wherein the detection comprises determining the amount of RNA transcript fragmentation in the sample relative to the amount of the first amplification product based on the average amount of the second amplification product and the third amplification product, wherein the comparison of the average amount of the second amplification product and the third amplification product relative to the first amplification product indicates the amount of RNA transcript fragmentation.

6. The method according to any one of claims 1-5, wherein the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, or at least 10 nucleotides.

7. The method according to any one of claims 1-5, wherein the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, or at least 100 nucleotides.

8. The method according to any one of claims 1-7, wherein the sample comprises RNA from a first compartment, a second compartment, or both.

9. The method according to any one of claims 1-8, wherein the first RNA sequence and the second RNA sequence are the same RNA transcript, but are present in both the first compartment and the second compartment.

10. The method according to any one of claims 8-9, wherein The first compartment contains the first RNA sequence and the second RNA sequence, and / or The second compartment contains the first RNA sequence and the second RNA sequence.

11. The method according to any one of claims 8-10, further comprising determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and the second compartment by measuring the amount or extent of a first or second RNA fragment of the first RNA sequence and the second RNA sequence in the first compartment and / or the second compartment.

12. The method of claim 11, wherein determining the difference between the amount of fragmentation of the RNA transcript in the first compartment and the second compartment comprises comparing the amount of fragmentation of the RNA transcript in the first compartment with the amount of fragmentation of the RNA transcript in the second compartment.

13. The method according to claims 8-12, further comprising one or more additional compartments.

14. The method of claim 13, wherein the one or more additional compartments comprise the first RNA sequence and the second RNA sequence and / or the third RNA sequence.

15. The method according to any one of claims 8-14, wherein the first compartment comprises RNA derived from a plurality of cells (e.g., cell compartments), a plurality of extracellular vesicles (EVs) / exosomes (e.g., EV compartments), nuclear RNA (e.g., nuclear compartments), cytoplasmic RNA (e.g., cytoplasmic compartments), mitochondrial RNA (e.g., mitochondrial compartments), chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

16. The method according to any one of claims 8-15, wherein the second compartment comprises RNA derived from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

17. The method according to any one of claims 8-16, wherein: The first compartment, the second compartment, or both contain RNA derived from a control sample; or The first compartment contains RNA derived from a control sample containing multiple cells, and the second compartment contains RNA derived from a test sample that is suspected to be fragmented, wherein the RNA derived from the test sample is selected from: multiple extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

18. The method according to any one of claims 8-17, wherein the first compartment, the second compartment, or both comprise RNA derived from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples.

19. The method of claim 18, wherein the environmental sample is selected from: water, wastewater, surface swabs, and air samples.

20. The method according to any one of claims 8-19, wherein the sample comprises RNA derived from a plurality of cells, RNA derived from a plurality of extracellular vesicles / exosomes, or both.

21. The method according to any one of claims 8-20, wherein: The first RNA sequence, the second RNA sequence, or both are derived from the RNA transcript; and the first and second RNA fragments of the second RNA sequence are fragments of the first RNA sequence.

22. The method of claim 21, wherein all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

23. The method of claim 21 or 22, wherein the RNA transcript is considered to be expressed in both the first compartment and the second compartment, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment.

24. The method of claim 23, wherein the first compartment comprises a plurality of cells and the second compartment comprises a plurality of extracellular vesicles / exosomes.

25. The method of claim 24, wherein, based in part on RNA sequencing data from the plurality of cells and RNA sequencing data from the plurality of extracellular vesicles / exosomes, the RNA transcript is believed to be expressed in both the first compartment containing the plurality of cells and the second compartment containing the plurality of extracellular vesicles.

26. The method of claim 24, wherein the first RNA sequence is expressed in a first compartment containing a plurality of cells and a second compartment containing a plurality of extracellular vesicles.

27. The method according to any one of claims 25-26, wherein the amount of the first amplification product from the first compartment comprising a plurality of cells is greater than the amount of the first amplification product from the second compartment comprising a plurality of extracellular vesicles.

28. The method according to any one of claims 24-27, wherein the first RNA fragment and the second RNA fragment in the second RNA sequence are expressed in a first compartment containing a plurality of cells and a second compartment containing a plurality of extracellular vesicles.

29. The method according to any one of claims 25-28, wherein the amount of the second amplification product from the second compartment comprising a plurality of extracellular vesicles is higher than the amount of the first amplification product from the second compartment comprising a plurality of extracellular vesicles.

30. The method according to any one of claims 22-29, wherein the first primer pair is capable of hybridizing and amplifying: the first RNA sequence of the RNA transcript from the first compartment comprising a plurality of cells and the second compartment comprising a plurality of extracellular vesicles / exosomes.

31. The method according to any one of claims 22-30, wherein the second primer pair is capable of hybridizing and amplifying: the first RNA fragment of the second RNA sequence from the first compartment comprising a plurality of cells and the second compartment comprising a plurality of extracellular vesicles / exosomes.

32. The method according to any one of claims 22-26, wherein the third primer pair is capable of hybridizing and amplifying: the second RNA fragment of the second RNA sequence from the first compartment comprising a plurality of cells and the second compartment comprising a plurality of extracellular vesicles / exosomes.

33. The method according to any one of claims 23-32, wherein the separation between the first RNA fragment and the second RNA fragment in the second RNA sequence is reflected in the RNA sequencing data from the second compartment because the number of sequencing reads mapped to the nucleotides in the second RNA sequence that separate the first RNA fragment and the second RNA fragment is reduced compared to the number of sequencing reads mapped to the corresponding nucleotides of the first RNA sequence in the first compartment.

34. The method according to any one of claims 1-33, wherein the RNA transcript is non-coding RNA.

35. The method of claim 34, wherein the non-coding RNA is selected from γ-RNA, snRNA, ncRNA, snoRNA, snaRNA, tRNA, rRNA, scRNA, telomerase RNA, fornix RNA, guide RNA, miRNA, antisense RNA, piRNA, and lncRNA.

36. The method according to any one of claims 1-35, further comprising selecting the first primer pair based on RNA sequencing data of the first RNA sequence from the first compartment.

37. The method of claim 36, wherein RNA sequencing data from the first RNA sequence in the first compartment show that the first RNA sequence is fragmented in at least a plurality of parts.

38. The method according to any one of claims 1-37, further comprising selecting the second primer pair based on RNA sequencing data from the second compartment.

39. The method of claim 38, wherein RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment.

40. The method according to any one of claims 4-39, further comprising selecting the third primer pair based on RNA sequencing data from the second compartment.

41. The method according to any one of claims 4-40, further comprising selecting at least a fourth primer pair, at least a fifth primer pair, at least a sixth primer pair, at least a seventh primer pair, at least an eighth primer pair, at least a ninth primer pair, or at least a tenth primer pair based on RNA sequencing data from the first compartment, the second compartment, or both.

42. The method of claim 41, wherein RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into a first RNA fragment and a second RNA fragment.

43. The method according to any one of claims 23-41, wherein the RNA sequencing data is whole transcriptome RNA sequencing data, exome RNA sequencing data, targeted RNA sequencing data, small RNA sequencing, and miRNA sequencing.

44. The method according to any one of claims 1-43, further comprising isolating RNA from the sample, wherein the RNA comprises the first RNA sequence and the second RNA sequence.

45. The method according to any one of claims 1-44, wherein fragmentation of the second RNA sequence occurs in the sample prior to isolating the RNA from the sample.

46. ​​The method according to any one of claims 1-45, wherein the fragmentation of the second RNA sequence is not the result of sonication, enzymatic fragmentation, chemical modification, UV irradiation, or thermal degradation.

47. The method according to any one of claims 1-46, wherein the fragmentation of the second RNA sequence is at least in part due to the presence of an exonuclease, an endonuclease, an RNA processing enzyme, an RNA-binding protein that binds to the second RNA sequence, or a combination thereof.

48. The method according to any one of claims 1-47, wherein the length of the first amplification product ranges from 50 to 220 nucleotides.

49. The method according to any one of claims 1-48, wherein the second amplification product, the third amplification product, or both have a length of at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, or at least 70 nucleotides; or the second amplification product, the third amplification product, or both have a length of no more than 75 nucleotides, no more than 70 nucleotides, or no more than 67 nucleotides.

50. The method according to any one of claims 1-49, wherein the length of the second amplification product, the third amplification product, or both ranges from 20 to 80 nucleotides.

51. The method according to any one of claims 1-45, wherein prior to the amplification, the method comprises reverse transcribing the first RNA sequence and the first RNA fragment into complementary DNA (cDNA) in a polymerase chain reaction (PCR).

52. The method of claim 46, wherein the first RNA sequence and the first RNA fragment are transcribed into complementary DNA (cDNA).

53. The method according to any one of claims 1-52, wherein the first primer pair comprises: (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:

22.

54. The method according to any one of claims 1-53, wherein the second primer pair comprises: (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:

21.

55. The method according to any one of claims 1-54, wherein the primer in the first primer pair, the primer in the second primer pair, or the primer in the third primer pair comprises one or more modified nucleotides.

56. The method of claim 55, wherein the modified nucleotide is a locked nucleotide.

57. A method for designing primers for detecting RNA transcript fragmentation, the method comprising: analyze RNA sequencing data from RNA collected in the first compartment, and RNA sequencing data from RNA collected in the second compartment. in The RNA sequencing data from the first compartment contains at most partially fragmented first RNA sequences; The RNA sequencing data from the second compartment includes the first RNA sequence, wherein all or part of the first RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment; and The first RNA sequence, the first RNA fragment, and the second RNA fragment are present in both the first compartment and the second compartment; and choose A first primer pair capable of hybridizing with and amplifying the first RNA sequence, and A second primer pair capable of hybridizing with the first RNA fragment.

58. A method for designing primers for detecting fragmentation of RNA transcripts, the method comprising: analyze RNA sequencing data from RNA collected in the first compartment, and RNA sequencing data of RNA collected from the second compartment; in The RNA sequencing data from the first compartment contains at most partially fragmented first RNA sequences; The RNA sequencing data from the second compartment includes a first RNA sequence, wherein all or part of the first RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment; and The first RNA sequence, the first RNA fragment, and the second RNA fragment are present in both the first compartment and the second compartment; and choose A first primer pair capable of hybridizing with and amplifying the first RNA sequence; A second primer pair capable of hybridizing with and amplifying the first RNA fragment; and A third primer pair capable of hybridizing with and amplifying the second RNA fragment.

59. The method of claim 57 or 58, wherein the first compartment comprises RNA derived from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof.

60. The method according to any one of claims 57-59, wherein the second compartment comprises RNA derived from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

61. The method according to any one of claims 57-60, wherein the first compartment, the second compartment, or both comprise RNA derived from a control sample.

62. The method according to any one of claims 57-61, wherein the first compartment, the second compartment, or both comprise RNA derived from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples.

63. The method of claim 62, wherein the environmental sample comprises: water, wastewater, surface swabs, and air samples.

64. A method for detecting fragmentation of RNA transcripts in a sample, the method comprising: The RNA transcript was reverse transcribed into complementary DNA (cDNA); Add a first primer pair and a second primer pair to the sample, wherein: The sample contains: The inverse complementary sequence of the first RNA sequence of the RNA transcript is fragmented at most partially to produce the inverse complementary sequence of the first RNA sequence. The inverse complementary sequence of the second RNA sequence of the RNA transcript in the sample, wherein all or part of the second RNA sequence is fragmented into at least a first RNA fragment and an optional second RNA fragment; The first primer pair is capable of hybridizing with the inverse complementary sequence of the first RNA sequence and amplifying all or part of the inverse complementary sequence of the first RNA sequence; The second primer pair can hybridize with the reverse complementary sequence of the first RNA fragment and amplify the reverse complementary sequence of the first RNA fragment; The first and second primer pairs are used to amplify the inverse complementary sequence of the first RNA sequence and the inverse complementary sequence of the first RNA fragment, thereby generating a first amplification product containing the inverse complementary sequence of the first RNA sequence and a second amplification product containing the inverse complementary sequence of the first RNA fragment. Quantify the amounts of the first amplification product and the second amplification product; and The fragmentation of the RNA transcript in the sample is detected based on the ratio of the amount of the second amplification product to the amount of the first amplification product.

65. The method of claim 64, wherein the first primer pair and the second primer pair are added to the sample in a separate reaction.

66. The method according to any one of claims 64-65, wherein after adding the first primer pair and the second primer pair to the sample but before the amplification, the method includes dividing the sample into a plurality of droplets.

67. The method according to any one of claims 64-65, wherein the method further comprises diluting the cDNA before adding the first primer pair and the second primer pair to the sample.

68. The method of claim 66, wherein the cDNA is diluted in water.

69. The method according to any one of claims 66-67, wherein the dilution range is from 1:2 to 1:

50.

70. The method according to any one of claims 66-69, further comprising: A third primer pair is added to the sample, wherein the third primer pair is capable of hybridizing with and amplifying the second RNA fragment; The second RNA fragment is amplified using the third primer pair in a reaction different from the first RNA sequence and the first RNA fragment, thereby producing a third amplification product containing the second RNA fragment or its reverse complementary sequence; Quantitative: The amount of the third amplification product; and The fragmentation of the RNA transcript in the sample can be detected based on one or more of the following: The ratio of the first amplification product, the second amplification product, to the third amplification product; The ratio of the first amplification product to the second amplification product; The ratio of the first amplification product to the third amplification product; The ratio of the first amplification product to the average amount of the second and third amplification products; The ratio of the second amplification product to the first amplification product; The ratio of the second amplification product, the third amplification product, and the first amplification product; The ratio of the third amplification product to the first amplification product; and The ratio of the average amount of the second amplification product and the third amplification product to the first amplification product.

71. The method according to any one of claims 65-70, wherein the sample is divided into a plurality of droplets, comprising: The first set of droplets, each droplet containing the reverse complementary sequence (cDNA) of the first RNA sequence, the first primer pair, and one or more reagents; The second set of droplets, each droplet containing the reverse complementary sequence of the first RNA fragment, the second primer pair, and one or more reagents; Optionally, a third group of droplets, each droplet containing the reverse complementary sequence of the second RNA fragment, a third primer pair, and one or more reagents; and / or A subset of droplets that does not contain the reverse complementary sequence of the first RNA sequence, the reverse complementary sequence of the first RNA fragment, and / or the reverse complementary sequence of the second RNA fragment.

72. The method according to any one of claims 70-71, wherein the method further comprises: The second amplicon product and / or the third amplicon product are adjusted by subtracting the amount of the second amplicon product from the first amplicon product (second amplicon product – first amplicon product) and / or subtracting the amount of the third amplicon product from the first amplicon product (third amplicon product – first amplicon product); and The detection of fragmentation of the RNA transcript in the sample is based on one or more of the following: The ratio of the first amplification product derived from multiple cells, the regulated second amplification product derived from multiple cells, to the regulated third amplification product derived from multiple cells; The ratio of the first amplification product derived from multiple extracellular vesicles / exosomes, the modified second amplification product derived from multiple extracellular vesicles / exosomes, to the modified third amplification product derived from multiple extracellular vesicles / exosomes; The ratio of the first amplification product derived from multiple cells to the regulated second amplification product derived from multiple cells; The ratio of the first amplification product derived from multiple extracellular vesicles / exosomes to the regulated second amplification product derived from multiple extracellular vesicles / exosomes; The ratio of the first amplification product derived from multiple cells to the regulated third amplification product derived from multiple cells; The ratio of the first amplification product derived from multiple extracellular vesicles / exosomes to the regulated third amplification product derived from multiple extracellular vesicles / exosomes; The ratio of the first amplification product derived from multiple cells to the average amount of the regulated second amplification product derived from multiple cells and the regulated third amplification product derived from multiple cells; as well as The ratio of the average amount of the first amplification product derived from multiple extracellular vesicles / exosomes to the average amount of the regulated second amplification product derived from multiple cells and the regulated third amplification product derived from multiple extracellular vesicles / exosomes. The ratio of the regulated second amplification product derived from multiple cells, the regulated third amplification product derived from multiple cells, and the first amplification product derived from multiple cells; The ratio of the regulated second amplification product derived from multiple extracellular vesicles / exosomes, the regulated third amplification product derived from multiple extracellular vesicles / exosomes, and the first amplification product derived from multiple extracellular vesicles / exosomes; The ratio of the regulated second amplification product derived from multiple cells to the first amplification product derived from multiple cells; The ratio of the regulated second amplification product derived from multiple extracellular vesicles / exosomes to the first amplification product derived from multiple extracellular vesicles / exosomes; The ratio of the regulated third amplification product derived from multiple cells to the first amplification product derived from multiple cells; The ratio of the regulated third amplification product derived from multiple extracellular vesicles / exosomes to the first amplification product derived from multiple extracellular vesicles / exosomes; The ratio of the average amount of the regulated second amplification product derived from multiple cells and the regulated third amplification product derived from multiple cells to the first amplification product derived from multiple cells; as well as The ratio of the average amount of the regulated second amplification product derived from multiple extracellular vesicles / exosomes and the regulated third amplification product derived from multiple extracellular vesicles / exosomes to the first amplification product derived from multiple extracellular vesicles / exosomes.

73. The method according to any one of claims 64-72, wherein the first RNA sequence and the second RNA sequence are derived from the same RNA transcript, but are present in both the first compartment and the second compartment.

74. The method according to any one of claims 64-73, wherein the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, or at least 10 nucleotides.

75. The method according to any one of claims 64-73, wherein the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a spacer of at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, or at least 100 nucleotides.

76. The method according to any one of claims 64-75, wherein the sample comprises RNA from a first compartment, a second compartment, or both.

77. The method according to any one of claims 72-77, further comprising determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and the second compartment.

78. The method of claim 77, wherein determining the difference between the amount of fragmentation of the RNA transcript in the first compartment and the second compartment comprises determining the ratio of the amount of fragmentation of the RNA transcript in the first compartment to the amount of fragmentation of the RNA transcript in the second compartment; or the ratio of the amount of fragmentation of the RNA transcript in the second compartment to the amount of fragmentation of the RNA transcript in the first compartment.

79. The method according to any one of claims 76-78, further comprising one or more additional compartments.

80. The method of claim 79, wherein the one or more additional compartments comprise a third RNA sequence.

81. The method according to any one of claims 76-80, wherein the first compartment comprises RNA derived from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof.

82. The method according to any one of claims 76-81, wherein the second compartment comprises RNA derived from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof.

83. The method according to any one of claims 76-82, wherein the first compartment, the second compartment, or both comprise RNA derived from a control sample comprising a plurality of cells and a test sample comprising a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

84. The method according to any one of claims 72-84, wherein the first compartment, the second compartment, or both comprise RNA derived from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetus, plant, and environmental samples.

85. The method of claim 84, wherein the environmental sample is selected from: water, wastewater, surface swabs, and air samples.

86. The method according to any one of claims 64-85, wherein the sample comprises RNA derived from a plurality of cells, RNA derived from a plurality of extracellular vesicles / exosomes, or both.

87. The method according to any one of claims 64-86, wherein the first RNA sequence, the second RNA sequence, or both are derived from the RNA transcript or the same RNA transcript.

88. The method of claim 87, wherein all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

89. The method of claim 88, wherein the RNA transcript is considered to be expressed in both the first compartment and the second compartment, based in part on RNA sequencing data from the first compartment and RNA sequencing data from the second compartment.

90. The method of claim 89, wherein the first compartment comprises a plurality of cells and the second compartment comprises a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum or combinations thereof.

91. The method of claim 90, wherein, based in part on RNA sequencing data of the plurality of cells and RNA sequencing data of the plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof, the RNA transcript is believed to be expressed in a first compartment comprising the plurality of cells and a second compartment comprising the plurality of extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or combinations thereof.

92. The method of claim 90, wherein the first RNA sequence is expressed in a first compartment comprising a plurality of cells and a second compartment comprising a plurality of extracellular vesicles.

93. The method according to any one of claims 91-92, wherein the amount of the first amplification product from the first compartment comprising a plurality of cells is greater than the amount of the first amplification product from the second compartment comprising a plurality of extracellular vesicles.

94. The method according to any one of claims 91-93, wherein the first RNA fragment and the second RNA fragment in the second RNA sequence are expressed in a first compartment containing a plurality of cells and a second compartment containing a plurality of extracellular vesicles.

95. The method according to any one of claims 91-94, wherein the amount of the second amplification product from the second compartment comprising a plurality of extracellular vesicles is higher than the amount of the first amplification product from the second compartment comprising a plurality of extracellular vesicles.

96. The method according to any one of claims 88-95, wherein the first primer pair is capable of hybridizing and amplifying: the first RNA sequence of the RNA transcript from the first compartment comprising a plurality of cells and the second compartment comprising a plurality of extracellular vesicles / exosomes.

97. The method according to any one of claims 88-96, wherein the second primer pair is capable of hybridizing and amplifying: the first RNA fragment of the second RNA sequence from the first compartment comprising a plurality of cells and the second compartment comprising a plurality of extracellular vesicles / exosomes.

98. The method of claim 97, wherein the ratio of the first compartment to the second compartment is determined using the following formula: Δcopy number （第一区室） =Amount of the second amplification product – Amount of the first amplification product; Δcopy number （第二区室） =Amount of the second amplification product – Amount of the first amplification product; X （第一区室） = ;as well as X （第二区室） = 。 99. The method of claim 98, wherein the detection of fragmentation of the RNA transcript in the sample is further based on a second ratio, wherein the second ratio is determined based on the following formula: Second ratio = Or the second ratio = .

100. The method of claim 99, wherein the second ratio is not 1.

101. The method according to any one of claims 91-100, wherein the RNA transcript is believed to be expressed in the plurality of cells and the plurality of extracellular vesicles / exosomes, based in part on RNA sequencing data from a plurality of cells and RNA sequencing data from a plurality of extracellular vesicles / exosomes.

102. The method according to any one of claims 88-101, wherein the separation between the first RNA fragment and the second RNA fragment in the second RNA sequence is reflected in the RNA sequencing data from the second compartment because the number of sequencing reads mapped to the nucleotides in the second RNA sequence that separate the first RNA fragment and the second RNA fragment is reduced compared to the number of sequencing reads mapped to the corresponding nucleotides in the first RNA sequence in the first compartment.

103. The method according to any one of claims 64-103, wherein the RNA transcript is non-coding RNA.

104. The method according to claim 103, wherein the non-coding RNA is selected from γ-RNA, snRNA, ncRNA, snoRNA, snaRNA, tRNA, rRNA, scRNA, telomerase RNA, fornix RNA, guide RNA, miRNA, antisense RNA, piRNA, and lncRNA.

105. The method according to any one of claims 88-104, further comprising selecting the first primer pair based on RNA sequencing data of the first RNA sequence from the first compartment.

106. The method of claim 105, wherein RNA sequencing data from the first RNA sequence in the first compartment show that the first RNA sequence is fragmented in at most a portion.

107. The method according to any one of claims 88-106, further comprising selecting the second primer pair based on RNA sequencing data from the second compartment.

108. The method of claim 107, wherein RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into the first RNA fragment and the second RNA fragment.

109. The method according to any one of claims 88-108, further comprising selecting the third primer pair based on RNA sequencing data from the second compartment.

110. The method of claim 109, further comprising selecting at least a fourth primer pair, at least a fifth primer pair, at least a sixth primer pair, at least a seventh primer pair, at least an eighth primer pair, at least a ninth primer pair, or at least a tenth primer pair based on RNA sequencing data from the first compartment, the second compartment, or both.

111. The method of claim 110, wherein RNA sequencing data from the second compartment show that the second RNA sequence is fragmented into the first RNA fragment and the second RNA fragment.

112. The method according to any one of claims 89-111, wherein the RNA sequencing data is whole transcriptome RNA sequencing data, exome RNA sequencing data, targeted RNA sequencing data, small RNA sequencing, and miRNA sequencing.

113. The method according to any one of claims 64-112, further comprising isolating RNA from the sample, wherein the RNA comprises the first RNA sequence and the second RNA sequence.

114. The method according to any one of claims 64-113, wherein fragmentation of the second RNA sequence occurs in the sample prior to isolating the RNA from the sample.

115. The method according to any one of claims 64-114, wherein the fragmentation of the second RNA sequence is not the result of sonication, enzymatic fragmentation, chemical modification, UV irradiation, or thermal degradation.

116. The method according to any one of claims 64-115, wherein the fragmentation of the second RNA sequence is at least in part due to the presence of an exonuclease, an endonuclease, an RNA processing enzyme, an RNA-binding protein that binds to the second RNA sequence, or a combination thereof.

117. The method according to any one of claims 64-116, wherein the length of the first amplification product ranges from 50 to 220 nucleotides.

118. The method according to any one of claims 64-117, wherein the second amplification product, the third amplification product, or both have a length of up to 90 nucleotides, up to 80 nucleotides, up to 70 nucleotides, up to 60 nucleotides, up to 50 nucleotides, up to 40 nucleotides, up to 30 nucleotides, up to 20 nucleotides, or up to 10 nucleotides.

119. The method according to any one of claims 64-118, wherein the second amplification product, the third amplification product, or both are at least 30 nucleotides in length.

120. The method according to any one of claims 64-119, wherein the first primer pair comprises: (ix) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (x) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; (xi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (xii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (xiii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; (xiv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; (xv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (xvi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:

22.

121. The method according to any one of claims 64-120, wherein the second primer pair comprises: (ix) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; (x) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (xi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (xii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (xiii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; (xiv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; (xv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (xvi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:

21.

122. The method according to any one of claims 64-121, wherein the primer in the first primer pair, the primer in the second primer pair, or the primer in the third primer pair comprises one or more modified nucleotides.

123. The method of claim 122, wherein the modified nucleotide is a locked nucleotide.

124. A primer set contains: A first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most a portion; and A second primer pair capable of hybridizing with and amplifying the first RNA fragment of the second RNA sequence. The second RNA sequence is fragmented, either entirely or partially, into at least a first RNA fragment and a second RNA fragment.

125. A primer set contains: A first primer pair capable of hybridizing with and amplifying a first RNA sequence, wherein the first RNA sequence is fragmented in at most part; A second primer pair capable of hybridizing with and amplifying the first RNA fragment of the second RNA sequence; and A third primer pair capable of hybridizing with and amplifying the second RNA fragment of the second RNA sequence. The second RNA sequence is fragmented, either entirely or partially, into at least a first RNA fragment and a second RNA fragment.

126. A primer set contains: The first primer pair contains (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22; The second primer pair contains (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:

21.

127. The primer set according to any one of claims 124-126, wherein the primers in the first primer pair and the primers in the second primer pair each contain one or more modified nucleotides.

128. The primer set according to claim 127, wherein the modified nucleotide is a locked nucleotide.

129. A kit for determining the structure of RNA transcripts; said kit comprising: The first primer pair contains (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22; The second primer pair contains (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21; and Instructions for performing the method according to any one of claims 1-123.

130. A kit for determining the structure of RNA transcription; said kit comprising: The first primer pair contains (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 5 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 7; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 11 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 12; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 15; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 22; The second primer pair contains (i) a forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 2; (ii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 3 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 4; (iii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 6 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 8; (iv) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 9 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 10; (v) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 13 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 14; (vi) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 16 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 17; (vii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 18 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 19; or (viii) A forward primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 20 and a reverse primer having a sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 21; and Instructions for performing the method according to any one of claims 1-123.

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