RNA fragmentation assay

Abstract

Provided herein are methods of detecting fragmentation of an RNA transcript in a sample where the method includes using a first primer pair to amplify a first RNA sequence of the RNA transcript that is at most partially fragmented and a second primer pair to amplify a second RNA sequence of the RNA transcript where all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a optionally second RNA fragment.

Description

RNA FRAGMENTATION ASSAY BACKGROUND

[0001] Traditional methods for detecting and characterizing fragmentation of an RNA transcript such as RNA-seq only provide average measurements of RNA fragments in a population of cells and do not quantify differences in fragmentation patterns. Therefore, techniques are needed to overcome these limitations and provide a more accurate, quantitative, and comprehensive understanding of RNA fragmentation. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith 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. SUMMARY

[0003] This disclosure features methods and compositions for detecting fragmentation of an RNA transcript in a sample. Understanding the structure of RNA transcripts, including whether they are fragmented or not, is valuable for several reasons. Firstly, the structure of an RNA molecule greatly influences its function within the cell. Full- length, unfragmented transcripts can be translated into functional proteins, while fragments may not code for complete proteins or may not be translated at all.Secondly, fragmentation can be a regulatory mechanism that cells use to control gene expression, either by destabilizing certain mRNAs to reduce their expression or by producing fragments that have distinct roles, such as regulatory non-coding RNAs. Thirdly, changes in mRNA fragmentation patterns can be indicative of various cellular conditions, including stress responses, diseases like cancer, or neurodegenerative disorders. Therefore, a comprehensive understanding of RNA fragmentation can provide insights into cellular function, gene regulation, and the mechanisms of disease.

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

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

[0006] EVs produced in tissues are released from the cells and find their way into the bloodstream. Liquid biopsies are thus an excellent source of EVs which could monitor patient health in clinical diagnostic tests. Cells grown in vitro also produce EVs and release them into the culture media. EVs isolated from culture media are a well- accepted model system to study EV biology.

[0007] Current EV RNA research focuses primarily on miRNAs which, as a class, have regulatory effects on biological pathways and can be involved in cell-to-cell communication. Many of these studies used miRNA-specific reagents for RNA isolation and RNA-Seq library preparation; this likely limits the findings of non- miRNA transcripts.

[0008] This disclosure features studies 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. The present findings show that the EV transcriptome is much different than the cellular transcriptome with key differences in the transcript structure of many non-coding small RNAs. This work provides a “Transcript Fragmentation Assessment PCR Assay” that can distinguish EV RNAs from cellular RNAs based on transcript structure differences using both quantitative PCR and droplet digital PCR (ddPCR). Such assays enable quantificationof EV-specific transcripts, which may eventually serve as biomarkers that monitor human health.

[0009] Aspects of the present disclosure include a method of detecting fragmentation of an RNA transcript in a sample, the method comprising: adding to the sample a first primer pair and a second primer pair, wherein the sample comprises: a first RNA sequence of the RNA transcript that is at most partially fragmented; a second RNA sequence of the RNA transcript, wherein all or a portion 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 to and amplifying all or a portion of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the first RNA fragment; amplifying the first RNA sequence and the first RNA fragment using the first and second primer pairs, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence and a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment; quantifying the amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein the amount of the second amplified product indicates RNA transcript fragmentation.

[0010] In some embodiments, the first primer pair and second primer pair are added to the sample in separate reactions.

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

[0012] In some embodiments, detecting comprises determining the amount of fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein a comparison between the amount of the second amplified product and the amount of the first amplified product indicates the amount or degree of RNA transcript fragmentation.

[0013] Aspects of the present disclosure includes a method of detecting fragmentation of an RNA transcript in a sample, the method comprising: adding to the sample at least a first primer pair, a second primer pair, and a third primer pair, wherein thesample comprises: a first RNA sequence of the RNA transcript that is at most partially fragmented; and a second RNA sequence of the RNA transcript, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and an optional a second RNA fragment, the first primer pair is capable of hybridizing to and amplifying all or a portion of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the first RNA fragment; and the third primer pair is capable of hybridizing to and amplifying the second RNA fragment; amplifying the first RNA sequence, the first RNA fragment, and the second RNA fragment using the first, second, and third primer pairs, respectively, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence, a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment, and a third amplified product comprising the sequence or reverse complement thereof of the second RNA fragment; quantifying: the amount of the first amplified product, the amount of the second amplified product; and the amount of the third amplified product; and detecting fragmentation of the RNA transcript in the sample based on the average amount of the second amplified product and the third amplified product, wherein the average amount of the second amplified product and the third amplified product indicates RNA transcript fragmentation.

[0014] In some embodiments, detecting comprises determining the amount of fragmentation of the RNA transcript in the sample relative to the amount of the first amplified product is based on the average amount of the second amplified product and the third amplified product, wherein a comparison between the average amount of the second amplified product and the third amplified product relative to the first amplified product indicates the amount of RNA transcript fragmentation.

[0015] In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a gap 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 gap 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, atleast 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 embodiments, the sample comprises RNA from a first compartment, a second compartment, or both.

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

[0018] In some embodiments, the the first compartment comprises the first RNA sequence and the second RNA sequence, and / or the second compartment comprises the first RNA sequence and the second RNA sequence.

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

[0020] In some embodiments, the first compartment comprises RNA originating from a plurality of cells (e.g., cellular compartment), a plurality of extracellular vesicles (EVs) / exosomes (e.g., EV compartment), nuclear RNA (e.g., nuclear compartment), cytoplasmic RNA (e.g., cytoplasmic compartment), mitochondrial RNA (e.g., mitochondrial compartment), chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the second compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from a control sample; or the first compartment comprises RNAoriginating from a control sample comprising a plurality of cells, and a second compartment comprises RNA originating from a test sample suspected to be fragmented, wherein the RNA originating from the test sample is selected from: a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, a fetus, a plant, and an environmental sample.

[0021] In some embodiments, the environmental sample is selected from: water, wastewater, surface swabs, and air samples. In some embodiments, the sample comprises RNA originating from a plurality of cells, RNA originating from a plurality of extracellular vesicles / exosomes, or both.

[0022] In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from the 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, the RNA transcript is deemed 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. In some embodiments, the first compartment comprises a plurality of cells and the second compartment comprises a plurality of extracellular vesicles / exosomes. In some embodiments, the RNA transcript is deemed to be expressed in both the first compartment comprising plurality of cells and the second compartment comprising the plurality of extracellular vesicles based in part on RNA sequencing data from the plurality of cells and RNA- sequencing data from the plurality of extracellular vesicles / exosomes. In some embodiments, the first RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles. In some embodiments, the amount of the first amplified product from the first compartment comprising the plurality of cells is more than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles. In some embodiments, the first RNA fragment and second RNA fragment in the second RNA sequence is expressed in the firstcompartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles. In some embodiments, the amount of the second amplified product from the second compartment comprising the plurality of extracellular vesicles is higher than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

[0023] In some embodiments, the first primer pair is capable of hybridizing to 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. In some embodiments, the second primer pair is capable of hybridizing to 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. In some embodiments, a third primer pair is capable of hybridizing to 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.

[0024] In some embodiments, a 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 as a reduction in the number of sequencing reads mapping to the nucleotides in the second RNA sequence separating the first RNA fragment and the second RNA fragment as compared to the number sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment. In some embodiments, the RNA transcript is a non-coding RNA.

[0025] In some embodiments, the non-coding RNA is selected from a Y-RNA, a snRNA, a ncRNA, a snoRNA, a snaRNA, a tRNA, a rRNA, a scRNA, a telomerase RNA, a vault RNA, a guide RNA, a miRNA, an antisense RNA, piRNA, and a IncRNA.In some embodiments, the method further comprises selecting the first primer pair based on RNA sequencing data for the first RNA sequence from the first compartment. In some embodiments, the RNA-sequencing data of the first RNA sequence from the first compartment shows the first RNA sequence is at most partially fragmented. In some embodiments, the method further comprises selectingthe second primer pair based on RNA sequencing data from the second compartment. In some embodiments, the RNA-sequencing data from the second compartment shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment. In some embodiments, the method further comprises selecting the third primer pair based on RNA sequencing data from the second compartment. In some embodiments, the method further comprises 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, selected based on RNA sequencing data from the first compartment, the second compartment, or both.

[0026] In some embodiments, RNA-sequencing data from the second compartment shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment. In some embodiments, the RNA-sequencing data is whole- transcriptome RNA sequencing data, exome RNA sequencing data, targeted RNA- sequencing data small RNA sequencing, and miRNA sequencing.

[0027] In some embodiments, the method further comprises isolating RNA from the sample wherein the RNA comprises the first RNA sequence and the second RNA sequence. The method of 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. In some embodiments, fragmenting of the second RNA sequence is not the result of sonication, tagmentation, chemical modification, UV irradiation, or heat degradation.

[0028] In some embodiments, the fragmentation of the second RNA sequence is due at least in part to the presence of exonucleases, endonucleases, RNA processing enzymes, an RNA binding protein bound to the second RNA sequence, or a combination thereof.In some embodiments, the first amplified product ranges from 50 to 220 nucleotides in length. In some embodiments, the second amplified product, the third amplified 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 amplified product, the third amplified product, or both have a length of no more than 75 nucleotides, no more than 70 nucleotides, or no more than 67nucleotides. In some embodiments, the second amplified product, the third amplified product, or both range from 20 to 80 nucleotides in length.

[0029] In some embodiments, before said amplifying, the method comprises reverse transcribing the first RNA sequence and the first RNA fragment into complementary DNA (cDNA) in a polymerase chain reaction (PCR). In some embodiments, transcribing the first RNA sequence and the first RNA fragment into complementary DNA (cDNA). In some embodiments, the first primer pair comprises: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22.

[0030] The method of any one of claims 1-53, wherein the second primer pair comprises a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence ofSEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

[0031] In some embodiments, the primers in the first primer pair, the primers in the second primer pair, or the primers in the third primer pair, comprise one or more modified nucleotides. In some embodiments, the modified nucleotide a locked nucleotide.

[0032] An aspect of the present disclosure includes a method for designing primers for detecting fragmentation of an RNA transcript, 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 comprises a first RNA sequence that is at most partially fragmented; the RNA sequencing data from the second compartment comprises the first RNA sequence, wherein all or a portion 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 that is capable of hybridizing to and amplifying the first RNA sequence, and a second primer pair that is capable of hybridizing to the first RNA fragment.

[0033] An aspect of the present disclosure includes a method for designing primers for detecting fragmentation of an RNA transcript, 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 comprises a first RNA sequence that is at most partially fragmented; the RNA sequencing data from the second compartment comprises the first RNA sequence, wherein all or a portion 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 that is capable of hybridizing to and amplifying the first RNA sequence; a second primer pair that is capable of hybridizing to and amplifying the first RNA fragment; and a third primer pair that is capable of hybridizing to and amplifying the second RNA fragment.

[0034] In some embodiments, the first compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof. In some embodiments, the second compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from a control sample. In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetuses, plants, and environmental samples. In some embodiments, the environmental samples comprises: water, wastewater, surface swabs, and air samples.

[0035] An aspect of the present disclosure includes a method of detecting fragmentation of an RNA transcript in a sample, the method comprising: reverse transcribing the RNA transcript into complementary DNA (cDNA); adding to the sample a first primer pair and a second primer pair, wherein: the sample comprises: a reverse complement of a first RNA sequence of the RNA transcript that is at most partially fragmented to produce a reverse complement of the first RNA sequence; a reverse complement of a second RNA sequence of the RNA transcript in the sample,wherein all or a portion 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 to and amplifying all or a portion of the reverse complement of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the reverse complement of the first RNA fragment; amplifying the reverse compliment of the first RNA sequence and the reverse complement of the first RNA fragment using the first and second primer pairs, thereby generating a first amplified product comprising the reverse complement of the first RNA sequence and a second amplified product comprising the reverse complement of the first RNA fragment; quantifying the amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on a ratio of the amount of the second amplified product and the amount of the first amplified product.

[0036] In some embodiments, the first primer pair and the second primer pair are added to the sample in separate reactions. In some embodiments, after adding to the sample the first primer pair and the second primer pair but before said amplifying, the method comprises partitioning the sample into a plurality of droplets.

[0037] In some embodiments the method further comprises, before adding to the sample the first primer pair and the second primer pair, diluting the cDNA. In some embodiments the cDNA is diluted in water. In some embodiments the the dilution ranges from 1:2 to 1 :50.

[0038] In some embodiments the method further comprises: adding to the sample a third primer pair, wherein the third primer pair is capable of hybridizing to and amplifying the second RNA fragment; amplifying the second RNA fragment using the third primer pair in a separate reaction from the first RNA sequence and the first RNA fragment, thereby generating a third amplified product comprising the sequence or reverse complement thereof of the second RNA fragment; quantifying: the amount of the third amplified product; and detecting fragmentation of the RNA transcript in the sample based on one or more of: a ratio of the first amplified product, the second amplified product, and the third amplified product; a ratio of the first amplified product and the second amplified product; a ratio of the first amplified product and the third amplified product; a ratio of the first amplified product and the average amount of the second amplified product and the third amplified product; a ratio of the second amplified product and the first amplified product; a ratio of the secondamplified product, the third amplified product, and the first amplified product; a ratio of the third amplified product and the first amplified product; and a ratio of the average amount of the second amplified product and the third amplified product, and the first amplified product.

[0039] In some embodiments the sample is partitioned into the plurality of droplets comprising: a first set of droplets, each droplet comprising the reverse complement of the first RNA sequence (cDNA), the first primer pair, and one or more reagents; a second set of droplets, each droplet comprising the reverse complement of the first RNA fragment, the second primer pair, and one or more reagents; optionally a third set of droplets, each droplet comprising the reverse complement of the second RNA fragment, a third primer pair, and one or more reagents; and / or a subset of droplets that do not comprise the reverse complement of the first RNA sequence, the reverse complement of the first RNA fragment, and / or the reverse complement of the second RNA fragment.

[0040] In some embodiments the method further comprises: adjusting the second amplicon product and / or the third amplicon product 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 detecting fragmentation of the RNA transcript in the sample is based on one or more of: a ratio of the first amplified product originating from a plurality of cells, the adjusted second amplified product originating from a plurality of cells, and the adjusted third amplified product originating from the plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes, the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes, and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the adjusted second amplified product originating from a plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and theadjusted third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the average amount of the adjusted second amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of cells; and a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the average amount of the adjusted second amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the adjusted second amplified product originating from a plurality of cells, the adjusted third amplified product originating from a plurality of cells, and the first amplified product originating from the plurality of cells; a ratio of the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes, the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes, and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the adjusted second amplified product originating from a plurality of cells and the first amplified product originating from a plurality of cells; a ratio of the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the adjusted third amplified product originating from a plurality of cells and the first amplified product originating from a plurality of cells; a ratio of the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the average amount of the adjusted second amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of cells, and the first amplified product originating from a plurality of cells; and a ratio of the average amount of the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes, and the first amplified product originating from a plurality of extracellular vesicles / exosomes.

[0041] In some embodiments, the first RNA sequence and the second RNA sequence are from the same RNA transcript but present in both a first compartment and a second compartment. In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a gap of at least 1nucleotide, 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 gap 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 comprises RNA from a first compartment, a second compartment, or both. In some embodiments the method further comprises determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment.

[0042] In some embodiments determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment comprises determining a ratio of the amount of the fragmentation of the RNA transcript in the first compartment and the amount of the fragmentation of the RNA transcript in the second compartment; or a ratio of the amount of the fragmentation of the RNA transcript in the second compartment and the amount of fragmentation of the RNA transcript in the first compartment. In some embodiments the method further comprises one or more additional compartments. In some embodiments the one or more additional compartments comprises a first RNA sequence, a first RNA fragment, a second RNA fragment, or a third RNA sequence.

[0043] In some embodiments, wherein the sample further comprises a reverse complement of a third RNA sequence of the RNA transcript that is at most partially fragmented to produce a reverse complement of the third RNA sequence. In some embodiments, wherein the sample further comprises a reverse complement of a fourth RNA sequence of the RNA transcript that is at most partially fragmented to produce a reverse complement of the third RNA sequence. In some embodiments, the sample comprises a reverse complement 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.

[0044] In some embodiments the first compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof. In some embodiments the second compartment comprising RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof. In some embodiments the first compartment, the second compartment, or both, comprise RNA originating 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, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0045] In some embodiments the first compartment, the second compartment, or both, comprise RNA originating from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, a fetus, a plant, and an environmental sample. In some embodiments the environmental sample is selected from: water, wastewater, surface swabs, and air samples. In some embodiments the sample comprises RNA originating from a plurality of cells, RNA originating from a plurality of extracellular vesicles / exosomes, or both. In some embodiments the first RNA sequence, the second RNA sequence, or both are: derived from the 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.

[0046] In some embodiments the RNA transcript is deemed 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.In some embodiments 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, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments the RNA transcript is deemed to be expressed in both the first compartment comprising plurality of cells and the second compartment comprising the plurality of extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof based in part on RNA sequencing data from the plurality of cells and RNA-sequencing data from theplurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.In some embodiments the first RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles.

[0047] In some embodiments the amount of the first amplified product from the first compartment comprising the plurality of cells is more than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles. In some embodiments the first RNA fragment and second RNA fragment in the second RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles. In some embodiments the amount of the second amplified product from the second compartment comprising the plurality of extracellular vesicles is higher than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

[0048] In some embodiments the first primer pair is capable of hybridizing to 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. In some embodiments the second primer pair is capable of hybridizing to 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.

[0049] In some embodiments the ratio for the first and second compartments is determined using the following formulas:Delta Copy Numb epfirst compartment) = the amount of the second amplified product - the amount of the first amplified product;Delta Copy Numbepsecond compartment) = the amount of the second amplified product - the amount of the first amplified product;X(first compartment)=Delta Copy Number(first compartment) originating from the plurality of cells amount of first amplified product in compartment originating from the plurality of cells ’ andX(second compartment) —Delta Copy Number(second compartment) originating from the plurality of extracellular vescicles / exosomes amount of first amplified product in compartment originating from the plurality of extracellular vescicles / exosomes

[0050] In some embodiments detecting fragmentation of the RNA transcript in the sample based is further based on a second ratio, wherein the second ratio is determined based on the following formula: second ratio =X(fi rstcompartment)Xfsecona compartment). . Xfsecond compartment) second ratio = — - - —Xffirst compartment)

[0051] In some embodiments the second ratio is not 1.

[0052] In some embodiments the RNA transcript is deemed to be expressed in both the plurality of cells and the plurality of extracellular vesicles based in part on RNA sequencing data from the plurality of cells and RNA-sequencing data from the plurality of extracellular vesicles / exosomes. In some embodiments a 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 as a reduction in the number of sequencing reads mapping to the nucleotides in the second RNA sequence separating the first RNA fragment and the second RNA fragment as compared to the number sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.

[0053] In some embodiments the RNA transcript is a non-coding RNA. In some embodiments the non-coding RNA is selected from a Y-RNA, a snRNA, a ncRNA, a snoRNA, a snaRNA, a tRNA, a rRNA, a scRNA, a telomerase RNA, a vault RNA, a guide RNA, a miRNA, an antisense RNA, piRNA, and a IncRNA.

[0054] In some embodiments the method further comprises selecting the first primer pair based on RNA sequencing data for the first RNA sequence from the first compartment. In some embodiments the RNA-sequencing data of the first RNA sequence from the first compartment shows the first RNA sequence is at most partially fragmented. In some embodiments the method further comprises selecting the second primer pair based on RNA sequencing data from the second compartment. In some embodiments RNA-sequencing data from the second compartment shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment.

[0055] In some embodiments the method further comprises selecting the third primer pair based on RNA sequencing data from the second compartment. In some embodiments the method further comprises 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 selected based on RNA sequencing data from the first compartment, the second compartment, or both.

[0056] In some embodiments RNA-sequencing data from the second compartment shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment. In some embodiments the RNA-sequencing data is whole- transcriptome RNA sequencing data, exome RNA sequencing data, targeted RNA- sequencing data small RNA sequencing, and miRNA sequencing. In some embodiments the method further comprises isolating RNA from the sample wherein the RNA comprises the first RNA sequence and the second RNA sequence. In some embodiments the fragmenting of the second RNA sequence occurs in the sample prior to isolating the RNA from the sample.

[0057] In some embodiments fragmenting of the second RNA sequence is not the result of sonication, tagmentation, chemical modification, UV irradiation, or heat degradation. In some embodiments fragmentation of the second RNA sequence is due at least in part to the presence of exonucleases, endonucleases, RNA processing enzymes, an RNA binding protein bound to the second RNA sequence, or a combination thereof. In some embodiments the first amplified product ranges from 50 to 220 nucleotides in length.

[0058] In some embodiments the second amplified product, the third amplified product, or both have a length of at most 90 nucleotides, at most 80 nucleotides, at most 70 nucleotides, at most 60 nucleotides, at most 50 nucleotides, at most 40 nucleotides, at most 30 nucleotides, at most 20 nucleotides, or at most 10 nucleotides. In some embodiments the second amplified product, the third amplified product, or both are at least 30 nucleotides in length. In some embodiments the first primer pair comprises: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence ofSEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22.

[0059] The method of any one of claims 64-120, wherein the second primer pair comprises: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primerhaving a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

[0060] In some embodiments the primers in the first primer pair, the primers in the second primer pair, or the primers in the third primer pair, comprise one or more modified nucleotides. In some embodiments the modified nucleotide a locked nucleotide.

[0061] An aspect of the present disclosure includes a composition comprising a set of primers, comprising: a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence, wherein the first RNA sequence is at most partially fragmented; and a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0062] An aspect of the present disclosure includes a composition comprising a set of primers, comprising: a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence wherein the first RNA sequence is at most partially fragmented; a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair that is capable of hybridizing to and amplifying a second RNA fragment of a second RNA sequence, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0063] An aspect of the present disclosure includes a composition comprising a set of primers, comprising: a first primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having asequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

[0064] In some embodiments, the primers in the first primer pair, the primers in the second primer pair, both comprise one or more modified nucleotides. In some embodiments, the modified nucleotide is a locked nucleotide.

[0065] An aspect of the present disclosure includes a kit for determining RNA transcription structure; the kit comprising: a first primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with atleast 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21; and instructions for performing any of the methods of the present disclosure.

[0066] An aspect of the present disclosure includes a kit for determining RNA transcription structure; the kit comprising: a first primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primerhaving a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22 a second primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21; and instructions for performing any of the methods of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0068] FIG. 1A illustrates a flow chart showing the steps of EV RNA isolation of cellular RNA isolation for preparing the RNA Seq libraries using qPCR described in Example 1.

[0069] FIG. IB illustrates a flow chart showing the steps for preparing the RNA-seq libraries for qPCR after EV and cellular RNA is isolated described in Example 1.

[0070] FIG. 1C illustrates a flow chart showing the RNA isolation steps that are common among samples prepared for quantitative PCR (qPCR) and droplet digital PCR (ddPCR) described in Example 1.

[0071] FIG. ID illustrates a flow chart showing steps required for qPCR versus ddPCR following SingleShot cell lysis.

[0072] FIG. IE illustrates a flow chart showing the steps required for qPCR following SingleShot cell lysis.

[0073] FIG. IF illustrates a flow chart showing the steps required for ddPCR following SingleShot cell lysis.

[0074] FIGs. 2A-2B shows estimate of maximal RFU of amplification traces (FIG. 2A) and baseline threshold values at 25% and 75% of the maximal RFU (FIG. 2B).

[0075] FIGs. 3A-3B shows example Bioanalyzer traces analyzing a cellular RNA- Seq library (FIG. 4A) or an EV RNA-Seq library (FIG. 4B).

[0076] FIG. 4 shows visualization and analysis of target gene reads on UCSC genome browser.

[0077] FIG. 5A shows transcript structure of RNY1 in cellular RNA and extracellular vesicle (EV) RNA. FIG. 5A shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNY 1 gene. The RNY 1 gene is 113 bp and is represented by the blue bar. In cellular RNA the RNY1 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNY1 transcript shows reads at the 5’ end and at the 3’ end but relatively few reads in the region between them. This data suggests that in EVs the RNY1 transcript is fragmented into a 5’ piece and a 3’ piece.

[0078] FIG. 5B shows transcript structure of RNY3 in cellular RNA and EV RNA. In particular, FIG. 5B shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNY3 gene. The RNY3 gene is 102 bp and is represented by the blue bar. In cellular RNA the RNY3 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNY3transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RNY3 transcript is fragmented into a 5’ piece and a 3’ piece.

[0079] FIG. 5C shows transcript structure of RNY4 in cellular RNA and EV RNA. FIG. 5C shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNY4 gene. The RNY4 gene is 96 bp and is represented by the blue bar. In cellular RNA the RNY4 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNY4 transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RNY4 transcript is fragmented into a 5’ piece and a 3’ piece.

[0080] FIG. 5D (SEQ ID NO: 29) shows transcript structure of RNY5 in cellular RNA and EV RNA by transcript fragmentation assessment PCR. FIG. 5D shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNY5 gene. The RNY5 gene is 84 bp and is represented by the blue bar. In cellular RNA the RNY5 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNY5 transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RNY5 transcript is fragmented into a 5’ piece and a 3’ piece.

[0081] FIG. 5E shows transcript structure of RMRP in cellular RNA and EV RNA. FIG. 5E shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RMRP gene. The RMRP gene is 264 bp and is represented by the blue bar. In cellular RNA the RMRP transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RMRP transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RMRP transcript is fragmented into a 5’ piece and a 3’ piece.

[0082] FIG. 5F shows transcript structure of RNU5B-1 in cellular RNA and EV RNA. FIG. 5F RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell linesare visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNU5B-1 gene. The RNU5B-1 gene is 116 bp and is represented by the green bar. In cellular RNA the RNU5B-1 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNU5B-1 transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RNU5B-1 transcript is fragmented into a 5’ piece and a 3’ piece.

[0083] FIG. 5G shows transcript structure of RNU5D-1 in cellular RNA and EV RNA. FIG. 5G shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNU5D-1 gene. The RNU5D-1 gene is 116 bp and is represented by the green bar. In cellular RNA the RNU5D-1 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNU5D-1 transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RNU5D-1 transcript is fragmented into a 5’ piece and a 3’ piece.

[0084] FIG. 5H shows transcript structure of RNU5E-1 in cellular RNA and EV RNA. FIG. 5H shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNU5E-1 gene. The RNU5E-1 gene is 120 bp and is represented by the blue bar. In cellular RNA the RNU5E-1 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNU5E-1 transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the RNU5E-1 transcript is fragmented into a 5’ piece and a 3’ piece.

[0085] FIG. 51 (SEQ ID NO: 30) shows transcript structure of SNORA15 in cellular RNA and EV RNA. FIG. 51 shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the SNORA15 gene. The SNORA15 gene is 133 bp and is represented by the blue bar. In cellular RNA the SNORA15 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVsthe SN0RA15 transcript shows reads at the 5’ end and at the 3’ end but fewer reads in the region between them. This data suggests that in EVs the SNORA15 transcript is fragmented into a 5’ piece and a 3’ piece.

[0086] FIG. 5J shows transcript structure of RNU12 in cellular RNA and EV RNA. FIG. 5 J shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the RNU12 gene. The RNU12 gene is 150 bp and is represented by the blue bar. In cellular RNA the RNU12 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the RNU12 transcript shows reads in the middle portion of the transcript, but significantly fewer reads at the 5’ end and 3’ ends. This data suggests that in EVs the RNU12 transcript is fragmented and primarily exists as a middle portion of the gene lacking the 5’ and 3’ ends.

[0087] FIG. 5K shows transcript structure of SCARNA10 in cellular RNA and EV RNA. FIG. 5K shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the SCARNA10 gene. The SCARNA10 gene is 330 bp and is represented by the blue bar. In cellular RNA the SCARNA10 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs 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 in EVs the SCARNA10 transcript is fragmented and primarily exists as a middle portion of the gene lacking the 5’ and 3’ ends.

[0088] FIG. 5L shows transcript structure of SCARNA5 in cellular RNA and EV RNA. FIG. 5L shows RNA-Seq results of RNA samples isolated from cellular RNA (first six tracks in red box) or EV RNA (next six tracks in green box) from several cell lines are visualized as custom tracks on the UCSC Genome Browser in the vicinity of the SCARNA5 gene. The SCARNA5 gene is 276 bp and is represented by the blue bar. In cellular RNA the SCARNA5 transcript is intact as the read pattern encompasses the entire coding region with no breaks. In contrast, in EVs the SCARNA5 transcript shows reads in the middle portion of the transcript, but significantly fewer reads at the 5’ end and 3’ ends. In addition, the middle portion of the SCARNA 5 transcript is further split with reads at the 5’ and 3’ ends, but fewer inthe middle. This data suggests that in EVs the SCARNA5 transcript is fragmented and primarily exists as a middle portion of the gene lacking the 5’ and 3’ ends. The middle portion is further fragmented into a 5’ piece and a 3’ piece.

[0089] FIGs. 6A-6D shows analysis of RNY5 by transcript fragmentation assessment PCR. FIG. 6A (SEQ ID NO: 29) shows RNA-Seq data of cellular and EV RNA isolated from several cell lines shows the transcript fragmentation pattern of EV RNA. FIG. 6B shows Transcript Fragmentation Assessment PCR control and test assay primer placement. FIG. 6C (SEQ ID Nos.: 1-4) shows sequences of the PCR primers. FIG. 6D shows transcript fragmentation assessment PCR results indicate that in all cell lines the RNY5 transcript is highly fragmented in EV RNA relative to cellular RNA with DDCq (used interchangeably as "delta-deltaCq” “DDCq” or “A ACq” ) scores ranging from -7.41 to -12.84.

[0090] FIG. 7A-7D shows analysis of RNU12 by transcript fragmentation assessment PCR. FIG. 7A shows RNA-Seq data of cellular and EV RNA isolated from several cell lines shows the transcript fragmentation pattern of EV RNA. The height of the EV tracks was custom set to show details of primer placement. FIG. 7B shows Transcript Fragmentation Assessment PCR control and test assay primer placement. FIG. 7C (SEQ ID Nos.: 5-8) shows sequences of the PCR primers. FIG. 7D Transcript fragmentation assessment PCR results indicate that in all cell lines the RNU12 transcript is highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -5.40 to -12.42.

[0091] FIGs. 8A-8D shows analysis of SCARNA5 by transcript fragmentation assessment PCR. FIG. 8A shows RNA-Seq data of cellular and EV RNA isolated from several cell lines shows the transcript fragmentation pattern of EV RNA. FIG. 8B shows Transcript Fragmentation Assessment PCR control and test assay primer placement. FIG. 8C (SEQ ID NOs: 9-12) shows sequences of the PCR primers. FIG. 8D shows transcript fragmentation assessment PCR results indicate that in all cell lines the SCARNA5 transcript is highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -5.96 to -8.75.

[0092] FIGs. 9A-9D shows analysis of SCARNA10 by transcript fragmentation assessment PCR. FIG. 9A RNA-Seq data of cellular and EV RNA isolated from several cell lines shows the transcript fragmentation pattern of EV RNA. FIG. 9B shows Transcript Fragmentation Assessment PCR control and test assay primer placement. FIG. 9C (SEQ ID NOs: 13-15) shows sequences of the PCR primers.FIG. 9D shows transcript fragmentation assessment PCR results indicate that in all cell lines the SCARNA10 transcript is highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -7.23 to -10.55.

[0093] FIGs. 10A-10D shows analysis of RMRP by transcript fragmentation assessment PCR. FIG. 10A shows RNA-Seq data of cellular and EV RNA isolated from several cell lines shows the transcript fragmentation pattern of EV RNA. FIG. 10B shows Transcript Fragmentation Assessment PCR control and test assay primer placement. FIG. 10C (SEQ ID NOs: 16-19) shows sequences of the PCR primers. FIG. 10D shows Transcript fragmentation assessment PCR results indicate that in all cell lines except Jurkat the RMRP transcript is highly fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -7.75 to -14.69.

[0094] FIGs. 11A-11D shows analysis of RNY1 by transcript fragmentation assessment PCR. FIG. 11A shows RNA-Seq data of cellular and EV RNA isolated from several cell lines shows the transcript fragmentation pattern of EV RNA. FIG. 11B shows transcript Fragmentation Assessment PCR control and test assay primer placement. FIG. 11C (SEQ ID NOs: 20-22) shows Sequences of the PCR primers. FIG. 11D shows transcript fragmentation assessment PCR results indicate that in all cell lines except Jurkat the RNY 1 transcript is fragmented in EV RNA relative to cellular RNA with DDCq scores ranging from -1.93 to -4.83.

[0095] FIGs. 12A-12B shows RNY5 EV fragments identified by Northernanalysis and RNA-Seq. FIG. 12A shows a diagram from Chakrabortty et al. (RNA, 21 (11): 1966-1979 (2015), doi: 10.1261 / rna.053629.115) depicting the start (red) and end (blue) positions of RNY5 fragments isolated from EVs derived from BJ or K562 cells as determined by Northern analysis. FIG. 12B (SEQ ID NO: 23) shows RNA-Seq data depicts the RNY5 transcript structure found in Ntera-2 EVs. The start and stop positions determined by Chakrabortty et al. (RNA, 21 (11): 1966-1979 (2015), doi: 10.1261 / rna.053629.115) are indicated. The RNY5 transcript structure in EVs are generally consistent. Note: Tracking molecular weight by Northern blot is difficult and not precise. The inaccuracy is worse with larger size. This may account for the slight discrepancy in transcript fragment location.

[0096] FIGs. 13A-13E shows Y-RNA bases that interact with the Ro60 protein. FIG. 13A (SEQ ID NO: 24) shows secondary structure of Xenopus laevis Y3 RNA with bases that are critical for RO60 binding shown in pink (diagram from Stein et al. Cell, 121(4): 529-539 (2005)). FIGs. 13B-13E (SEQ ID NOs: 25-28) shows screenshots ofMCF7 exosomal RNA at the RNY1, RNY3, RNY4 and RNY5 locus. The RNY1 is in a 3’ to 5’ orientation. RNY3, RNY4 and RNY5 are in a 5’ to 3’ orientation. A red box encloses the bases critical for RO60 binding in the 5’ region of the transcript. A blue box encloses the bases critical for RO60 binding in the 3’ region of the transcript. The yellow oval highlights the only base that is not conserved in the sequence motifs.

[0097] FIG. 14 shows a comparison of the analysis of RMRP by transcript fragmentation assessment using qPCR as shown in FIGs 10A-10D with analysis of RMRP by transcript fragmentation assessment using ddPCR. Transcript fragmentation using ddPCR is determined by first calculating a “Delta Copy number”, 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 calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA).. The results of ddPCR in the A549 cell line show that the RMRP transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0098] FIG. 15 shows a comparison of the analysis of RNY5 by transcript fragmentation assessment using qPCR as shown in FIGs 6A-6D with analysis of RNY5 by transcript fragmentation assessment using ddPCR. Transcript fragmentation using ddPCR is determined by first calculating a “Delta Copy number”, 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 calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA). The results of ddPCR in the A549 cell line show that the RNY5 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0099] FIG. 16 shows a comparison of the analysis of RNU12 by transcript fragmentation assessment using qPCR as shown in FIGs 7A-7D with analysis of RNU12 by transcript fragmentation assessment using ddPCR. Transcript fragmentation using ddPCR is determined by first calculating a “Delta Copy number”, which is the amount of the second amplified product of the first fragment of thesecond RNA sequence minus the amount of the first amplified product of the first RNA sequence (e.g., “Average Control” - “Average Test”) and then calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA). The results of ddPCR in the A549 cell line show that the RNU12 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0100] FIG. 17 shows a comparison of the analysis of SCARNA5 by transcript fragmentation assessment using qPCR as shown in FIGs 8A-8D with analysis of SCARNA5 by transcript fragmentation assessment using ddPCR. Transcript fragmentation using ddPCR is determined by first calculating a “Delta Copy number”, 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 calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA). The results of ddPCR in the A549 cell line show that the SCARNA5 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0101] FIG. 18 shows a comparison of the analysis of SCARNA10 by transcript fragmentation assessment using qPCR as shown in FIGs 9A-9D with analysis of SCARNA10 by transcript fragmentation assessment using ddPCR. Transcript fragmentation using ddPCR is determined by first calculating a “Delta Copy number”, 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 calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA). The results of ddPCR in the A549 cell line show that the SCARNA10 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0102] FIG. 19 shows a comparison of the analysis of RNY1 by transcript fragmentation assessment using qPCR as shown in FIGs 11 A-l ID with analysis of RNY 1 by transcript fragmentation assessment using ddPCR. Transcript fragmentationusing ddPCR is determined by first calculating a “Delta Copy number”, 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 calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA). The results of ddPCR in the A549 cell line show that the RNY 1 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results. DETAILED DESCRIPTION5.1. Method of detecting fragmentation of an RNA transcript in a sample

[0103] This disclosure features a method of detecting fragmentation of an RNA transcript in a sample, where the method includes using a first primer pair and a second primer pair. The method includes adding to the sample a first primer pair and a second primer pair. In some embodiments, the first primer pair and second primer pair are added in separate reactions.

[0104] In some embodiments, the sample comprises: a first RNA sequence of the 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%) fragmented); and a second RNA sequence of the RNA transcript, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and an optional second RNA fragment. In some embodiments, the first RNA sequence is not fragmented. The first primer pair is capable of hybridizing to and amplifying all or a portion of the first RNA sequence, and the second primer pair is capable of hybridizing to and amplifying the first RNA fragment.

[0105] In some embodiments, the method includes amplifying the first RNA sequence and the first RNA fragment using the first and second primer pairs, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence and a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment. In some embodiments, the method also includes quantifying the amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on the amount of the second amplifiedproduct, wherein the amount of the second amplified product relative to the first amplified product indicates RNA transcript fragmentation.

[0106] In some embodiments, “at most partially fragmented” refers to at most 40% fragmented. In some embodiments, “at most partially fragmented” refers to at most 30% fragmented. In some embodiments, “at most partially fragmented” refers to at most 20% fragmented. In some embodiments, “at most partially fragmented” refers to at most 10% fragmented. In some embodiments, “at most partially fragmented” refers to at most 5% fragmented. In some embodiments, “at most partially fragmented” refers to at most 50% fragmented. In some embodiments, “at most partially fragmented” refers to at most 60% fragmented. In some embodiments, “at most partially fragmented” refers to at most 70% fragmented. In some embodiments, “at most partially fragmented” refers to at most 80% fragmented. In some embodiments, “at most partially fragmented” refers to at most 90% fragmented. In some embodiments, the first RNA sequence is more than 40% fragmented, more than 50% fragmented, more than 60% fragmented, more than 70% fragmented, more than 80% fragmented, or more than 90% fragmented. For example, in some embodiments, if there is an abundant amount of the first RNA sequence in cellular RNA compared to EV RNA, there may be a higher degree of fragmentation.

[0107] In some embodiments, detecting includes determining the amount of fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein a comparison between the amount of the second amplified product and the amount of the first amplified product acting as a test indicates the amount of RNA transcript fragmentation.

[0108] This disclosure features a method of detecting fragmentation of an RNA transcript in a sample, where the method includes using a first primer pair and a second primer pair. The method includes adding to the sample at least a first primer pair, a second primer pair, and a third primer pair. In some embodiments, the at least a first primer, at least a second primer pair, and at least a third primer pair are added in separate reactions. In some embodiments, the sample comprises: a first RNA sequence of the RNA transcript that is at most partially fragmented; and a second RNA sequence of the RNA transcript, wherein all or a portion 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 to and amplifying all ora portion of the first RNA sequence, the second primer pair is capable of hybridizing to and amplifying the first RNA fragment; and the third primer pair is capable of hybridizing to 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, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence, a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment, and a third amplified product comprising the sequence or reverse complement thereof of the second RNA fragment. The method also includes quantifying: the amount of the first amplified product, the amount of the second amplified product; and the amount of the third amplified product; and detecting fragmentation of the RNA transcript in the sample based on the average amount of the second amplified product and the third amplified product, wherein the average amount of the second amplified product and the third amplified product indicates RNA transcript fragmentation, and the amount of the first amplified product act as a test.

[0109] In some embodiments, detecting includes determining the amount of fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein a comparison between the amount of the second amplified product and the amount of the first amplified product indicates the amount of RNA transcript fragmentation.

[0110] In some embodiments, the sample comprises RNA from a first compartment, a second compartment, or both. In some embodiments, the first compartment comprises the first RNA sequence and the second compartment comprises the second RNA sequence or the first compartment comprises the second RNA sequence and the second compartment comprises the first RNA sequence, or the first compartment comprises the first RNA sequence and the second RNA sequence, or the second compartment comprises the first RNA sequence and the second RNA sequence.5.2. Method of detecting fragmentation of an RNA transcript in a sample comprising at least a first compartment and a second compartment[OHl] This disclosure features a method of detecting fragmentation of an RNA transcript in a sample, where the method includes using a first primer pair and a second primer pair. The method includes adding to the sample a first primer pair and a second primer pair. The sample comprises: a first compartment comprising a firstRNA sequence of the RNA transcript, wherein the first RNA sequence that is at most partially fragmented; and a second compartment comprising a second RNA sequence of the RNA transcript, where all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and an optionally second RNA fragment. The first primer pair is capable of hybridizing to and amplifying all or a portion of the first RNA sequence, and the second primer pair is capable of hybridizing to 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 generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence and a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment. The method also includes quantifying the amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein the amount of the second amplified product indicates RNA transcript fragmentation and the amount of the first amplified product acts as a test while the amount of the second amplified product acts as a control.

[0112] Compartmentalization is a kind of membrane-bound cellular organization that divides the internal volume of the cell into discrete (but often interconnected) compartments. These compartments each have distinct properties and functions. For example, a cellular compartment can include closed parts within the cytosol of a cell, surrounded by a single or double lipid layer membrane.

[0113] In some embodiments, the sample comprises 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 present in both a first compartment and a second compartment. In some embodiments, the first compartment comprises the first RNA sequence and the second compartment comprises the second RNA sequence or the first compartment comprises the second RNA sequence and the second compartment comprises the first RNA sequence. In certain embodiments, the first compartment comprises the first RNA sequence and the second RNA sequence. In certain embodiments, the second compartment comprises the first RNA sequence and the second RNA sequence.

[0114] In some embodiments, the method includes determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment. In certain embodiments, the first RNA sequence may be present in similar levels as the second RNA fragment in some compartments, e.g., such as a compartment comprising a plurality of cells. In some embodiments, the first RNA sequence is present in different levels in a first compartment compared to a second compartment.

[0115] In some embodiments, determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment comprises a comparison between the amount of the fragmentation of the RNA transcript in the first compartment and the amount of the fragmentation of the RNA transcript in the second compartment.

[0116] In some embodiments, the first compartment comprises RNA originating from a plurality of cells (e.g., cellular compartment), a plurality of extracellular vesicles (EVs) / exosomes (e.g., EV compartment), nuclear RNA (e.g., nuclear compartment), cytoplasmic RNA (e.g., cytoplasmic compartment), mitochondrial RNA (e.g., mitochondrial compartment), chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In certain embodiments, the second compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0117] In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from a control sample. In some embodiments, the first compartment comprises RNA originating from a control sample comprising a plurality of cells, and a second compartment comprises RNA originating from a test sample suspected to be fragmented, wherein the RNA originating from the test sample is selected from: a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0118] In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, a fetus, a plant, and an environmental sample.

[0119] In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from the RNA transcript; and wherein the first and second RNA fragments of the second RNA sequence are fragments of the first RNA sequence.

[0120] In some embodiments, all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

[0121] In some embodiments, the RNA transcript is deemed 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.

[0122] In some embodiments, 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, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0123] In some embodiments, the RNA transcript is deemed to be expressed in both the first compartment comprising plurality of cells and the second compartment comprising the plurality of extracellular vesicles based in part on RNA sequencing data from the plurality of cells and RNA-sequencing data from the plurality of extracellular vesicles / exosomes.

[0124] In some embodiments, the first RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0125] In some embodiments, the amount of the first amplified product from the first compartment comprising the plurality of cells is more than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the amount of the first amplified product from the first compartment comprising the plurality of cells is similar to the amount of the second amplified product from the first compartment comprising the plurality of cells.

[0126] In some embodiments, the first RNA fragment and second RNA fragment in the second RNA sequence is expressed in the first compartment comprising theplurality of cells and the second compartment comprising the plurality of extracellular vesicles.

[0127] In some embodiments, the amount of the second amplified product from the second compartment comprising the plurality of extracellular vesicles is higher than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

[0128] Aspects of the present disclosure include a method of detecting fragmentation of an RNA transcript in a sample, where the method includes using a first primer pair and a second primer pair. The method includes adding to the sample at least a first primer pair, a second primer pair, and a third primer pair. The sample comprises: a first compartment comprising a first RNA sequence, where the first RNA sequence of the RNA transcript that is at most partially fragmented; and a second compartment comprising a second RNA sequence of the RNA transcript, wherein all or a portion 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 to and amplifying all or a portion of the first RNA sequence, the second primer pair is capable of hybridizing to and amplifying the first RNA fragment; and the third primer pair is capable of hybridizing to 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, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence, a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment, and a third amplified product comprising the sequence or reverse complement thereof of the second RNA fragment. The method also includes quantifying: the amount of the first amplified product, the amount of the second amplified product; and the amount of the third amplified product; and detecting fragmentation of the RNA transcript in the sample based on the average amount of the second amplified product and the third amplified product, wherein the average amount of the second amplified product and the third amplified product indicates RNA transcript fragmentation, and the amount of the first amplified product act as a test.

[0129] In some embodiments, the sample includes one or more additional compartments.

[0130] In some embodiments, the method includes isolating RNA from a first compartment, from a second compartment, one or more additional compartments. In some embodiments, the one or more additional compartments comprise a first RNA sequence, a first RNA fragment, and / or optionally a third RNA fragment.

[0131] In some embodiments, the first primer pair is capable of hybridizing to 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.

[0132] In some embodiments, the second primer pair is capable of hybridizing to 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.

[0133] In some embodiments, a third primer pair is capable of hybridizing to 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.5.3. RNA: source, transcripts, sequences

[0134] In some embodiments, the sample includes RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof or a combination thereof. In some embodiments, the sample includes RNA originating from a plurality of cells (e.g., cellular compartment), a plurality of extracellular vesicles (EVs) / exosomes (e.g., EV compartment), nuclear RNA (e.g., nuclear compartment), cytoplasmic RNA (e.g., cytoplasmic compartment), mitochondrial RNA (e.g., mitochondrial compartment), chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the sample includes RNA originating from a compartment comprising an animal cell. In some embodiments, the sample includes RNA originating from a compartment of a eukaryotic cell. In some embodiments, the sample includes RNA originating from a compartment of a plant cell.

[0135] In some embodiments, the sample includes RNA originating from a plurality of cells, RNA originating from a plurality of extracellular vesicles / exosomes, or both.

[0136] In some embodiments, the sample includes RNA originating from blood, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, a fetus, a plant, and / or an environmental sample.

[0137] In some embodiments, the sample includes RNA from a first compartment, a second compartment, or both. In one embodiment, the first compartment comprises the first RNA sequence and the second compartment comprises the second RNA sequence or the first compartment comprises the second RNA sequence and the second compartment comprises the first RNA sequence, or the first compartment comprises the first RNA sequence and the second RNA sequence, or the second compartment comprises the first RNA sequence and the second RNA sequence.

[0138] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA. In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment comprises RNA originating from a plurality of cells. In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment comprises RNA originating from a plurality of extracellular vesicles / exosomes.

[0139] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the second compartment comprising RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof. In some embodiments where the sample includes RNA from a first compartment and a second compartment, the second compartment comprising RNA originating from a plurality of cells. In some embodiments where the sample includes RNA from a first compartment and a second compartment, the second compartment comprising RNA originating from a plurality of extracellular vesicles / exosomes.

[0140] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof; and the second compartment comprising RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA,mitochondrial RNA, or a combination thereof. In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment comprises RNA originating from a plurality of cells; and the second compartment comprising RNA originating from a plurality of extracellular vesicles / exosomes.

[0141] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment, the second compartment, or both, comprise RNA originating from a control sample.

[0142] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment, the second compartment, or both, comprise RNA originating from blood, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, a fetus, a plant, and an environmental sample. In such embodiments, the environmental sample is selected from: water, wastewater, surface swabs, and air samples.

[0143] In some embodiments, the first RNA sequence, the second RNA sequence, or both are derived from the 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 present in both a first compartment and a second compartment.

[0144] In some embodiments where the sample includes RNA from a first compartment and a second compartment, all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

[0145] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the RNA transcript is deemed 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.

[0146] In some embodiments where the sample includes RNA from a first compartment and a second compartment, the first compartment comprises a plurality of cells and the second compartment comprises a plurality of extracellular vesicles / exosomes. In such embodiments, the RNA transcript is deemed to be expressed in both the plurality of cells and the plurality of extracellular vesicles basedin part on RNA sequencing data from the plurality of cells and RNA-sequencing data from the plurality of extracellular vesicles / exosomes.

[0147] In some embodiments, the RNA transcript is deemed to be expressed in both the first compartment comprising plurality of cells and the second compartment comprising the plurality of extracellular vesicles based in part on RNA sequencing data from the plurality of cells and RNA-sequencing data from the plurality of extracellular vesicles / exosomes.

[0148] In some embodiments, the RNA transcript is a non-coding RNA. In some embodiments, the non-coding RNA is selected from: a Y-RNA, a snRNA, a ncRNA, a snoRNA, a snaRNA, a tRNA, a rRNA, a scRNA, a telomerase RNA, a vault RNA, a guide RNA, a miRNA, an antisense RNA, piRNA, and a IncRNA.

[0149] In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a gap 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.

[0150] In some embodiments, the first RNA fragment and the second RNA fragment are separated in the second RNA sequence by a gap 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.

[0151] In some embodiments, a separation (a gap) 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 as a reduction in the number of sequencing reads mapping to the nucleotides in the second RNA sequence separating the first RNA fragment and the second RNA fragment as compared to the number sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.

[0152] In some embodiments, the method includes selecting the first primer pair based on RNA sequencing data for the first RNA sequence from the first compartment. In some embodiments, the RNA-sequencing data of the first RNA sequence from the first compartment shows the first RNA sequence is at mostpartially fragmented. In such cases, the state of fragmentation of the first RNA sequence is factored into selection of the first primer pair.

[0153] In some embodiments, the method includes selecting the second primer pair based on RNA sequencing data from the second compartment. In some embodiments, the RNA-sequencing data from the second compartment shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment. In such cases, the fragmentation of the second RNA sequence is factored into selection of the second primer pair.

[0154] In some embodiments, the method includes selecting the third primer pair based on RNA sequencing data from the second compartment. For example, the RNA-sequencing data from the second compartment shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment. In such cases, the fragmentation of the second RNA sequence is factored into selection of the third primer pair.

[0155] In some embodiments, the method includes selecting at least fourth primer pair, at least 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 selected based on RNA sequencing data from the first compartment, the 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.

[0156] In some embodiments, the RNA-sequencing data is whole-transcriptome RNA sequencing data, exome RNA sequencing data, targeted RNA-sequencing data small RNA sequencing, and miRNA sequencing. The RNA-sequencing data can be view on a genome viewer in order to visualize localization of the sequencing reads within the sequence of the RNA transcript. One non-limiting example of a genome viewer is the UCSC Genome Browser.5.4. Isolating, (not) fragmenting, and amplifying

[0157] In some embodiments, the method also includes isolating RNA from the sample where the RNA includes the first RNA sequence and the second RNA sequence.

[0158] In some embodiments, the method also includes isolating RNA from the sample where the sample includes a first compartment, a second compartment, or both, and RNA is isolated separately from each compartment. In some embodiments, the method also includes isolating RNA from the sample where the sample includes a first compartment, a second compartment, or both, and RNA is isolated from each compartment separately and then pooled. In some embodiments, the method also includes isolating RNA from the sample where the sample includes a first compartment, a second compartment, or both, and RNA is isolated from both compartments in the same solution.

[0159] In some embodiments, the method comprises culturing the cells prior to RNA isolation. In some embodiments, the method comprises precipitating EVs from suspended or adherent cells. In some embodiments, the method comprises isolation of EV RNA following precipitation. In some embodiments, the method comprises isolation of cellular RNA from from suspended or adherent cells. In some embodiments, the method comprises isolation of cellular RNA following precipitation.

[0160] In some embodiments, the fragmenting of the second RNA sequence occurs in the sample prior to isolating the RNA from the sample. In some embodiments, fragmenting of the second RNA sequence is not the result of sonication, tagmentation, chemical modification, UV irradiation, and / or heat degradation. In some embodiments, the method does not include a fragmenting step.

[0161] In some embodiments, the method comprises preparing the sample comprising the first RNA sequence and the second RNA sequence isolated from EVs. In some embodiments, the method comprises fragmentation of cellular RNA.

[0162] In some embodiments, the method comprises performing End Repair of EV and cellular RNA samples. In some embodiments, the method comprises performing poly(A) tailing on the EV and cellular RNA samples. In some embodiments, the method comprises running the EV and cellular RNA samples in a thermal cycler for continuous synthesis. In some embodiments, the method comprises purifying and concentrating the reverse complement (cDNA) of the EV and cellular RNA samples.

[0163] In some embodiments, the amplifying includes amplifying the RNA transcript (e.g., the first RNA sequence and the second RNA sequence) under conditions sufficient to allow amplification of the RNA transcript. Amplification of the RNAtranscript (e.g., the first RNA sequence and the second RNA sequence) can include a reverse transcription step, whereby the RNA is reverse transcribed into complementary DNA (cDNA). Amplification of the RNA transcript can include one or more of the following components: primers, which are short, single-stranded DNA sequences that are complementary to the regions flanking the target sequence in the template DNA or template RNA; polymerase (e.g., a DNA-dependent DNA polymerase or a RNA-dependent DNA polymerase), which are heat-stable DNA polymerase enzymes that synthesizes new strands of DNA using the primers and template DNA or template RNA; nucleotides (dNTPs), for example, a mix of deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, dGTP) used to supply the A, T, C, and G bases that the polymerase adds to the new DNA strand; and a buffer solution that provides conditions sufficient for the polymerase to function, where the buffer solution includes one or more of salt(s), a pH buffer, and magnesium.

[0164] In some embodiments, the first amplified product is 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 length. In some embodiments, the first amplified product has a nucleotide length ranging from 40 to 230 nucleotide base pairs in length. In some embodiments, the first amplified product has a nucleotide length ranging from 50 to 220 nucleotide base pairs in length. In some embodiments, the first amplified product has a nucleotide length ranging from 55 to 215 nucleotide base pairs in length. In some embodiments, the first amplified product is no more than 220 nucleotides in length, or no more than 215 nucleotides in length.

[0165] In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 90 nucleotides, at most 80 nucleotides, at most 70 nucleotides, at most 60 nucleotides, at most 50 nucleotides, at most 40 nucleotides, at most 30 nucleotides, at most 20 nucleotides, or at most 10 nucleotides. In some embodiments, the second amplified product, the third amplified product, orboth have a length of at most 90 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 80 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 70 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 60 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 50 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 40 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both have a length of at most 30 nucleotides. In some embodiments, the second amplified product, the third amplified product, or both range from 20 to 80 nucleotides in length.

[0166] In some embodiments, the second amplified product, the third amplified product, or both are at least 20 nucleotides in length. In some embodiments, the first amplified 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 amplified product, the third amplified product, or both are no more than 80 nucleotides in length or no more tan 70 nucleotides in length.

[0167] In some embodiments, the method further comprises determining the number of library amplification PCR cycles for each EV RNA and cellular sample. For example, in order to determine the number of amplification cycles for the libraries, the method comprises analyzing the first amplified product and the second amplified product using 2 different thresholds which effectively set lower and upper boundaries for the cycles during which exponential amplification is taking place. In some embodiments, the method comprises setting a baseline threshold of about 25% and 75% of the maximal RFU of the first amplicon product. In some embodiments, the method comprises collecting Cq values at 25% and 75% of the maximal RFU of the first amplicon product and the second amplified product.

[0168] In some embodiments, the method includes analyzing RNA-sequencing data with the aim of determining structure (e.g., fragmentation) of an RNA transcript. RNA-sequencing data can be derived from RNA isolated from the same RNA sample from which the methods described herein are performed. For example, RNA can beisolated from a plurality of cells and used to derive RNA-sequencing data as well as be subjected to the methods described herein (e.g., the Transcript Fragmentation Assessment PCR). Alternatively, RNA-sequencing data can be derived from RNA isolated from a different RNA sample from which the methods described herein are performed.

[0169] RNA-sequencing data can be generated from RNA-sequence libraries prepared using an RNA-sequence library preparation kit. Non-limiting examples of kits that can be used to generate a sequencing library include: an Illumina TruSeq RNA Library Prep Kit; a NEBNext Ultra II Directional RNA Library Prep Kit; a QIAseq Stranded Total RNA Lib Prep Kit; a Takara SMART-Seq v4 Ultra Low Input RNA Kit; a Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit; and a KAPA Stranded mRNA-Seq Kit.5.5. Reverse transcribing RNA transcript into complementary DNA (cDNA)

[0170] In some embodiments, the RNA transcript of the sample is reverse transcribed into cDNA. In certain embodiments, depending on the PCR reactions performed (e.g., quantitative PCR or droplet digital PCR), the first RNA sequence and first RNA fragment are reverse transcribed into cDNA prior to amplification.

[0171] In some embodiments where qPCR is performed, the method comprises reverse transcribing the first RNA sequence into cDNA and performing qPCR and amplification in the same reaction. In some embodiments where qPCR is performed, the method comprises reverse transcribing the 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 comprises performing cDNA conversion and qPCR in the same reaction. In some embodiments where qPCR is performed, the method comprises reverse transcribing the second RNA fragment into cDNA and performing qPCR in the same reaction. In some embodiments where qPCR is performed, the method comprises reverse transcribing the third RNA fragment into cDNA and performing qPCR in the same reaction. In some embodiments, the first primer pair and the second primer pair are added to the sample in separate reactions. In some embodiments, the method comprises adding a first primer pair to a reaction (e.g., first reaction) containing the sample. In some embodiments, the method comprises adding a second primer pair to a reaction (e.g., second reaction) containing the sample.

[0172] In some embodiments, where ddPCR is performed, the method comprises reverse transcribing the first RNA sequence into cDNA by mixing the first RNA sequence with a DNase master mix and running the sample with the DNase master mix in a thermocycler, followed by mixing the sample with a reverse transcription master mix, and running the sample using the corresponding protocol in a thermocycler. The resulting cDNA sample is then mixed with an EvaGreen Supermix. Thus, in some embodiments, the method comprises mixing the cDNA sample with an EvaGreen Supermix kit. In some embodiments, the method further comprises generating droplets from the sample. After droplet generation, the method comprises running the sample with the EvaGreen Supermix in a thermocycler. In some embodiments, when ddPCR is used for amplification, the cDNA is prepared in bulk.5.6. Primers, primer pairs, and sets of primers

[0173] This disclosure also features primers, primer pairs, and sets of primer pairs that can be used for detecting fragmentation of an RNA transcription in a sample.

[0174] In one embodiment, a first primer pair is capable of hybridizing to and amplifying a first RNA sequence, where the first RNA sequence is at most partially fragmented.

[0175] In another embodiment, a second primer pair is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence, where all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0176] In one embodiment, a set of primer pairs includes: a first primer pair is capable of hybridizing to and amplifying a first RNA sequence, where the first RNA sequence is at most partially fragmented; and a second primer pair is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence, where all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0177] In some embodiments, the method includes a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence, where the first RNA sequence is at most partially fragmented; and a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence, where all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0178] In some embodiments, a first primer pair is capable of hybridizing to and amplifying a first RNA sequence wherein the first RNA sequence is at most partially fragmented; a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair that is capable of hybridizing to and amplifying a second RNA fragment of a second RNA sequence, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0179] In some embodiments, the first primer pair is selected from: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; and a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22.

[0180] In some embodiments, the second primer pair is selected: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having asequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; and a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

[0181] In some embodiments, a set of primer pairs includes a first primer pair selected from: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having asequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; and a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22; and a second primer pair selected from: a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; and a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

[0182] Designing PCR primers that target (and enable amplification of) the fragmented transcripts found in a compartment (e.g., EVs) can be difficult because many fragments are small (under 30 bases) and unable to accommodate forward and reverse PCR primers.

[0183] In some embodiments, the PCR primers include modifications that increase the 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 (LNA), peptide nucleic acids (PNA)) and incorporation of one or more modified internucleoside linkages (e.g., phosphorothioate bonds).

[0184] In some embodiments, the PCR primers include modifications that facilitate 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 (LNA), peptide nucleic acids (PNA)) and incorporation of one or more modified internucleoside linkages (e.g., phosphorothioate bonds).

[0185] In some instances, modified oligonucleotides can be incorporated into a PCR primer to amplify a fragmented transcript that is otherwise difficult to amplify. Nonlimiting examples of modified oligonucleotides include a locked nucleic acid (LNA), a peptide nucleic acid (PNA), an oligonucleotide with a non-natural base (e.g., inosine), a cytosine analog (e.g., 5-methylcytosine), a thymine analog (5- bromouracil), an adenine analog (e.g., 2-aminoadenine), and a guanine analog).

[0186] In some embodiments, locked nucleic acid bases are incorporated into a PCR primer or pair of PCR primer to increase the primer Tm and improve PCR performance.5.7. Determining the amount of fragmentation of an RNA transcript5.7.1. Quantitative PCR (qPCR)

[0187] In some embodiments, the method includes quantifying the amount of the first amplified product and the amount of the second amplified product. In some embodiments, quantifying the amount of the first amplified product and the amount of the second amplified product is done using quantitative PCR. In quantitative PCR (qPCR), Delta quantification cycle (Cq) (ACq) is a calculation used to quantify the relative amount of target gene expression compared to a reference or “housekeeping” gene. The Cq is the cycle number at which the fluorescence generated within a reaction crosses the threshold, which is typically set within the exponential phase of the amplification plot. The Delta Cq is calculated by subtracting the Cq value of the housekeeping gene from the Cq value of the target gene in the same sample: ACq = Cq (target gene) - Cq (reference gene). A lower ACq value indicates a higher level of target gene expression, and a higher ACq value suggests a lower level of target gene expression. Using a housekeeping gene for normalization helps to account for variations in the amount or quality of starting material, efficiency of amplification,efficiency of reverse transcription, and differences in overall transcriptional activity between different samples or between different compartments (e.g., between a first compartment and a second compartment).

[0188] In some embodiments, to be certain that the variations in Cq values are due to real biological changes and not technical issues, the results (e.g., amounts of RNA) are normalized. Non-limiting examples of normalization methods include: “delta-Cq”, “Delta-Delta-Cq”, The Pfaffl method, or the Livak method. In such example, the Cq values of the sample are compared to the Cq values of several reference (housekeeping) genes.

[0189] The Delta-Delta Cq method makes the assumption that the amplification (PCR) efficiencies of the reference and target samples are almost 100% and within 5% of each other. The Livak method for relative quantification assumes PCR efficiencies of the target and reference genes should be between 90 and 100%. The Pfaffl method assumes different reaction efficiencies for the reference and target genes.

[0190] Selection of reference genes whose expression levels are not expected to change during the experiment is a consideration in obtaining reliable and accurate results. Non-limiting examples of housekeeping genes include actin, alpha-tubulin, GAPDH, and ubiquitin.

[0191] Notably, the Delta-Delta Cq method assumes that the amplification (PCR) efficiencies of the reference and target samples are almost 100% and within 5% of each other. Other normalization methods include the Delta-Cq Method and the Pfaffl Method.

[0192] In some embodiments, the method includes detecting fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein the amount of the second amplified product indicates RNA transcript fragmentation and the amount of the first amplified product act as a control. In one embodiment, the method includes detecting fragmentation of the RNA transcript in the sample based on the amount (ACq) of the second amplified product, wherein the amount of the second amplified product indicates RNA transcript fragmentation and the amount (ACq) of the first amplified product act as a test.

[0193] In some embodiments, the method includes detecting comprises determining the amount of fragmentation of the RNA transcript in the sample based on the average amount of the second amplified product and the third amplified product, wherein acomparison between the average amount average amount of the second amplified product and the third amplified product and the amount of the first amplified product acting as a test indicates the amount of RNA transcript fragmentation. For example, the method includes detecting comprises determining the amount (ACq) of fragmentation of the RNA transcript in the sample based on the average amount (ACq) of the second amplified product and the third amplified product, wherein a comparison between the average amount (ACq) of the second amplified product and the third amplified product and the amount (ACq) of the first amplified product acting as a test indicates the amount of RNA transcript fragmentation.

[0194] In some embodiments, the method includes determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment. In some embodiments, determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment comprises a comparison between the amount of the fragmentation of the RNA transcript in the first compartment and the amount of the fragmentation of the RNA transcript in the second compartment.5.7.1. Droplet digital PCR (ddPCR)

[0195] Aspects of the present disclosure include a method of detecting fragmentation of an RNA transcript in a sample using ddPCR. The method includes: reverse transcribing the RNA transcript into complementary DNA (cDNA); adding to the sample a first primer pair and a second primer pair, wherein: the sample comprises: a reverse complement of a first RNA sequence of the RNA transcript that is at most partially fragmented to produce a reverse complement of the first RNA sequence; a reverse complement of a second RNA sequence of the RNA transcript in the sample, wherein all or a portion 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 to and amplifying all or a portion of the reverse complement of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the reverse complement of the first RNA fragment; amplifying the reverse compliment of the first RNA sequence and the reverse complement of the first RNA fragment using the first and second primer pairs, thereby generating a first amplified product comprising the reverse complement of the first RNA sequence and a second amplified product comprising the reverse complement of the first RNA fragment; quantifyingthe amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on a ratio of the amount of the second amplified product and the amount of the first amplified product.

[0196] In some embodiments, the first primer pair and the second primer pair are added in separate reactions. In some embodiments, a test sample and a control sample can be included in the same reaction comprising a first primer pair or a second primer pair. In some embodiments, a first compartment and a second compartment can be included in the same reaction comprising a first primer pair or a second primer pair. In some embodiments, a test sample and a control sample can be included in different reactions. In certain embodiments, the test sample comprises a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, a control sample comprises RNA originating from a plurality of cells. In some embodiments, the control sample

[0197]

[0198] In some embodiments, the method includes quantifying the amount of the first amplified product and the amount of the second amplified product. In some embodiments, quantifying the amount of the first amplified product and the amount of the second amplified product is done using droplet digital PCR (ddPCR).

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

[0200] In some embodiments, when the sample is a cellular sample (e.g., cellular RNA that is reverse transcribed into cDNA), the cDNA is diluted prior to performing ddPCR. In some embodiments, the cDNA is diluted in water. In some embodiments, the dilution ranges from 1 :2 to 1 :50. In some embodiments, the dilution ranges from 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 comprises generating a plurality of droplets. In some embodiments, for a 20 pL reaction, the plurality of droplets ranges from at least15,000 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 droplets to 20,000 droplets. In some embodiments, the plurality of droplets ranges from 18,000 droplets to 20,000 droplets. In some embodiments, once the reaction mix is ready, the method comprises loading 20-22 pL of the reaction mixture into a sample well of a DG8 cartridge for QX200 Droplet Generator (Bio-Rad #1864008) followed by 70 pL of QX200 Droplet Generation Oil for EvaGreen® (Bio-Rad #1864005 or 1864006) into the oil wells, according to the QX200 Droplet Generator Instruction Manual (Bio-Rad #10031907), or for the automatic droplet generator, follow instructions in the Automated Droplet Generator Instruction Manual (Bio-Rad #10043138). In some embodiments, the sample is partitioned into the plurality of droplets comprising: a first set of droplets, each droplet comprising the reverse complement of the first RNA sequence (cDNA), the first primer pair, and one or more reagents; a second set of droplets, each droplet comprising the reverse complement of the first RNA fragment, the second primer pair, and one or more reagents; optionally a third set of droplets, each droplet comprising the reverse complement of the second RNA fragment, a third primer pair, and one or more reagents; and / or a subset of droplets that do not comprise the reverse complement of the first RNA sequence, the reverse complement of the first RNA fragment, and / or the reverse complement of the second RNA fragment.

[0202] In some embodiments, the method further comprises performing DNA fragmentation by restriction digestion prior to droplet generation. Restriction enzyme digestion can be carried out as a separate reaction before ddPCR reaction setup. After droplet generation with the QX200 droplet generator following manufacturer’s instructions, the method comprises running the sample through a thermal cycler and subsequent reading of droplets in the QX200 Droplet Reader (Bio-Rad) following manufacturer’s instructions. After thermal cycling, the method comprises analyzing the sample using a QX Manager software. The QX Manager software will perform data acquisition and analyze the droplets using the EvaGreen fluorescent dye. The concentration of the amplicon products (e.g., first amplicon product, second amplicon product, or both) is reported as number of copies / pL of the final lx ddPCR reaction.

[0203] In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the first amplified product, the second amplified product, and the third amplified product. In some embodiments,when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the first amplified product and the second amplified product. In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the first amplified product and the third amplified product. In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the first amplified product and the average amount of the second amplified product and the third amplified product. In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the second amplified product and the first amplified product. In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the second amplified product, the third amplified product, and the first amplified product. In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the third amplified product and the first amplified product. In some embodiments, when ddPCR is used, detecting fragmentation of the RNA transcript in the sample based on a ratio of the average amount of the second amplified product and the third amplified product, and the first amplified product.

[0204] In some embodiments, before detecting the ratio of the second amplified product and the first amplified product; the third amplified product and the first amplified product, or the average of the second and third amplified product and the first amplified product, the method comprises adjusting or calculating the second amplified product or third amplified product for the presence of the first amplified product by calculating a “delta copy number” of the second RNA sequence. The “delta copy number” of the second RNA sequence is the amount of e.g., a second amplified product or third amplified product from the second RNA sequence minus the amount of the first amplified product from the first RNA sequence. For example, as shown in FIGs. 14-19, the “delta copy number” of the second RNA sequence, is shown 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 comprises calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment) and for a EV RNA sample (e.g., second compartment), and a second ratio of X(ceiiuiar RNA) to X(EVRNA).

[0205] In some embodiments, detecting fragmentation of the RNA transcript in the sample based on one or more of: a ratio of the first amplified product originating from a plurality of cells, the second amplified product originating from a plurality of cells, and the third amplified product originating from the plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes, the second amplified product originating from a plurality of extracellular vesicles / exosomes, and the third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the second amplified product originating from a plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the second amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the third amplified product originating from a plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the average amount of the second amplified product originating from a plurality of cells and the third amplified product originating from a plurality of cells; and a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the average amount of the second amplified product originating from a plurality of cells and the third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the second amplified product originating from a plurality of cells, the third amplified product originating from a plurality of cells, and the first amplified product originating from the plurality of cells; a ratio of the second amplified product originating from a plurality of extracellular vesicles / exosomes, the third amplified product originating from a plurality of extracellular vesicles / exosomes, and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the second amplified product originating from a plurality of cells and the first amplified product originating from a plurality of cells; a ratio of the second amplified product originating from a plurality of extracellular vesicles / exosomes and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of thethird amplified product originating from a plurality of cells and the first amplified product originating from a plurality of cells; a ratio of the third amplified product originating from a plurality of extracellular vesicles / exosomes and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the average amount of the second amplified product originating from a plurality of cells and a third amplified product originating from a plurality of cells, and the first amplified product originating from a plurality of cells; and a ratio of the average amount of the second amplified product originating from a plurality of extracellular vesicles / exosomes and a third amplified product originating from a plurality of extracellular vesicles / exosomes, and the first amplified product originating from a plurality of extracellular vesicles / exosomes.

[0206] In some embodiments, the first RNA sequence and the second RNA sequence are from the same RNA transcript but present in both a first compartment and a second compartment.

[0207] In some embodiments, the sample comprises RNA from a first compartment, a second compartment, or both.

[0208] In some embodiments, the method further comprises determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment. In some embodiments, determining the difference between the amount of fragmentation of the RNA transcript between the first compartment and second compartment comprises determining a ratio of the amount of the fragmentation of the RNA transcript in the first compartment and the amount of the fragmentation of the RNA transcript in the second compartment; or a ratio of the amount of the fragmentation of the RNA transcript in the second compartment and the amount of fragmentation of the RNA transcript in the first compartment.

[0209] In some embodiments, the first compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof. In some embodiments, the second compartment comprising RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, or a combination thereof. In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from a control sample comprising a plurality of cells, and a test sample comprising a pluralityof extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. wherein all or part of the RNA transcript is expressed in the first compartment, the second compartment, or both.

[0210] In some embodiments, the RNA transcript is deemed 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.

[0211] In some embodiments, 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, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof. In some embodiments, the RNA transcript is deemed to be expressed in both the first compartment comprising plurality of cells and the second compartment comprising the plurality of extracellular vesicles, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof based in part on RNA sequencing data from the plurality of cells and RNA- sequencing data from the plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0212] In some embodiments, the first RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles.

[0213] In some embodiments, the amount of the first amplified product from the first compartment comprising the plurality of cells is more than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

[0214] In some embodiments, the first RNA fragment and second RNA fragment in the second RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles.

[0215] In some embodiments, the amount of the second amplified product from the second compartment comprising the plurality of extracellular vesicles is higher thanthe amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

[0216] In some embodiments, the first primer pair is capable of hybridizing to 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. In some embodiments, the second primer pair is capable of hybridizing to 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.

[0217] In some embodiments, detecting fragmentation of the RNA transcript, when using ddPCR, is determined by the ratio for the first and second compartments using the following formulas:Delta Copy Numb epfirst compartment) = the amount of the second amplified product - the amount of the first amplified product;Delta Copy Numbepsecond compartment) = the amount of the second amplified product - the amount of the first amplified product; v > Delta Copy Number(first compartment) originating from the plurality of cells , (first compartment) — ~ ; ; : : : : ~ ; 7 ~ amount of first amplified product in compartment originating from the plurality of ce ”lls ; andX(second compartment) —Delta Copy Number(second compartment) originating from the plurality of extracellular vescicles / exosomes amount of first amplified product in compartment originating from the plurality of extracellular vescicles / exosomes

[0218] In some embodiments, detecting fragmentation of the RNA transcript in the sample is further based on a second ratio, wherein the second ratio is determined . . . ,, .. . ,, . . Xffirst compartment) based on the following formula: second ratio = — - - - - .Xfsecond compartment)

[0219] In some embodiments, detecting fragmentation of the RNA transcript in the sample is further based on a second ratio, wherein the second ratio is determined . < < n . . Xfsecond compartment) based on the following formula: a second ratio = — - — ..Xfnrst compartment)

[0220] In some embodiments, the second ratio is not 1. For example, as shown inFIGs. 14-19, transcript fragmentation using ddPCR is determined by first adjusting the second amplified product or third amplified product for the presence of the first amplified product (e.g., in a case where a second primer pair hybridizes to the firstRNA fragment of the second RNA sequence, and a portion of the first RNA sequence). For example, the second and third amplified products are “corrected” by the first amplified product (subtracting the amount of first transcript from the amount of second, in cases where the second primer set amplifies the first RNA fragment and a portion of the first RNA sequence. In some embodiments, adjusting the second amplified product comprises subtracting the amount of the second amplified product from the amount of the first amplified product from the first RNA sequence to determine a “delta copy number”. In some embodiments, the method further comprises, after calculating the delta copy number for a first compartment and a second compartment, the method comprises calculating a first ratio (X) of the delta copy number of a second amplified product (e.g., “Avg Control” assay) and the amount of a first amplified product (e.g., “Average test” assay) for a cellular RNA sample (e.g., first compartment) and a EV RNA sample (e.g., second compartment), followed by a second ratio of Xceiiuiar RNA to XEV RNA. In some embodiments, the detecting fragmentation of the RNA transcript in the sample is based on the second ratio.

[0221] In some embodiments, a first amplified product includes the reverse complement of a first RNA sequence of an RNA transcript that is at most partially fragmented (e.g., “test” assay containing RNA regions that are expressed in both cellular and EV RNA compartments) and a second amplified product includes the reverse complement of a fragmented portion of the first RNA sequence (e.g., “control”).

[0222] In some embodiments, the RNA transcript is deemed to be expressed in both the plurality of cells and the plurality of extracellular vesicles based in part on RNA sequencing data from the plurality of cells and RNA-sequencing data from the plurality of extracellular vesicles / exosomes. In some embodiments, a 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 as a reduction in the number of sequencing reads mapping to the nucleotides in the second RNA sequence separating the first RNA fragment and the second RNA fragment as compared to the number sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.5.8. Designing primers for detecting fragmentation of an RNA transcript

[0223] This disclosure features a method for designing primers for detecting fragmentation of an RNA transcript in a sample. Designing PCR assays that target the fragmented transcripts found in a sample or in a particular compartment (e.g., EVs) can be difficult because many fragments are small (under 30 bases) and unable to accommodate forward and reverse PCR primers. The methods described herein enable design of primers that address these limitations.

[0224] The method includes analyzing: RNA-sequencing data of RNA collected from a first compartment, and RNA sequencing data of RNA collected from a second compartment. The RNA-sequencing data from the first compartment comprises a first RNA sequence that is at most partially fragmented, and the RNA sequencing data form the second compartment comprises a second RNA sequence, wherein all or a portion 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 present in both a first compartment and a second compartment. The method also includes selecting a first primer pair that is capable of hybridizing to and amplifying the first RNA sequence, and a second primer pair that is capable of hybridizing to the first RNA fragment.

[0225] This disclosure features a method for designing primers for detecting fragmentation of an RNA transcript. The method includes analyzing RNA-sequencing data of RNA collected from a first compartment, and RNA sequencing data of RNA collected from a second compartment. The RNA-sequencing data from the first compartment comprises a first RNA sequence that is at most partially fragmented; the RNA sequencing data form the second compartment comprises a second RNA sequence, wherein all or a portion 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 present in both the first compartment and the second compartment. The method includes selecting a first primer pair that is capable of hybridizing to and amplifying the first RNA sequence; a second primer pair that is capable of hybridizing to and amplifying the first RNA fragment; and a third primer pair that is capable of hybridizing to and amplifying the second RNA fragment.

[0226] In some embodiments, the first compartment comprises RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA,cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0227] In some embodiments, the second compartment comprising RNA originating from a plurality of cells, a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplasts, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

[0228] In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from a control sample.

[0229] In some embodiments, the first compartment, the second compartment, or both, comprise RNA originating from blood, plasma, liquid biopsy, tissue, saliva, urine, feces, cerebrospinal fluid, placenta, fetuses, plants, and environmental samples.

[0230] In some embodiments, environmental samples comprise: water, wastewater, surface swabs, and air samples.5.9. Kits

[0231] This disclosure features a kit for performing a method of detecting fragmentation of an RNA transcript in a sample. In one embodiment, the kit includes a set of primers, that includes: a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence, wherein the first RNA sequence is at most partially fragmented; and a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

[0232] In another embodiment, the kit includes a set of primers, that includes: a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence wherein the first RNA sequence is at most partially fragmented; a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair that is capable of hybridizing to and amplifying a second RNA fragment of a second RNA sequence, wherein all or a portion 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 set of primer, where the primer pairs are selected from: a first primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22; a second primer pair comprising a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10; a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14;a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17; a forward primerhaving a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21; and instructions for performing any of the methods described herein. EXAMPLES6.1. Example 1. Transcript Fragmentation Assessment PCR Assay

[0234] In this work, EV RNA was analyzed using RNA isolation and RNA-Seq library preparation reagents that assess the entire transcriptome, from small miRNAs to large mRNAs and non-coding RNAs. The disclosure presents findings showing that the EV transcriptome is much different than the cellular transcriptome with key differences in the transcript structure of many non-coding small RNAs. In this Example, data is provided for a “Transcript Fragmentation Assessment PCR Assay” that can distinguish EV RNAs from cellular RNAs based on transcript structure differences using qPCR or ddPCR. Such assays could quantify EV-specific transcripts, which may eventually serve as biomarkers that monitor human health.6.1.1. Methods6.1.1.1 Part 1: RNA Isolation6.1.1.1.1 Cell culture

[0235] NT era-2 clone DI was obtained from Millipore Sigma (Cat# 01071221-1VL).ATCC supplied the 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 culture conditions recommended by ATCC. Cells grown in suspension or adherent culture are appropriate for EV analysis by RNA-Seq. For cells grown in suspension culture 1.4 mL of cells at 70%-90% confluence is required for EV purification. For adherent cells, one well of a 6-well tissue culture plate at 70%-90% confluence is required for EV purification.

[0236] Cells are initially cultured under growth conditions recommended by the cell supplier or as previously determined. For example, cell culture media was changed to media containing exosome-free FBS (System Biosciences Innovation, Cat# EXO- FBSHI-50A-1) one to three days prior to harvest. At the time of harvest the cells were healthy and approximately 90% confluent. One to three days prior to EV harvest the cell culture media is changed to media containing exosome-depleted FBS (SBI). Thecells should be at a density such that they will be 70%-90% confluent at the time of harvest.6.1.1.1.2 Precipitation of EVs from suspension cells

[0237] For suspension cells grown in culture media containing exosome-depleted FBS for 1-3 days are 70%-90% confluent at the time of EV harvest, the following steps are performed for precipitation of EVs:

[0238] Collect 1.4 mL of suspension cells in a microcentrifuge tube.

[0239] As a negative control, collect 1.4 mL of culture media containing exosome- depleted FBS (no cells) in a microcentrifuge tube.

[0240] Centrifuge samples at 3,000 x g for 5 minutes in a microcentrifuge.

[0241] Transfer 1.2 mL of supernatant into a fresh microcentrifuge tube being careful not to transfer pelleted cells.

[0242] As an optional step, the tube containing pelleted cells can be saved for harvesting cellular RNA as directed in section 3a.

[0243] Centrifuge the 1.2 mL samples at 12k RPM for 15 minutes.

[0244] Transfer 1.0 mL of supernatant to a fresh microcentrifuge tube being careful not to transfer pelleted material. Store samples on ice.

[0245] Add 200 mL of ExoQuick TC Tissue Culture Media Exosome Precipitation Solution (SBI) to each sample. (Note: ExoQuick-TC is viscous and difficult to pipet. When working with this reagent pipet slowly using wide-bore pipet tips. Ensure that the entire 200 mL volume is added to each sample).

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

[0247] Store samples at 4°C for at least 18 hours to precipitate EVs.

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

[0249] Proceed to section “isolation of EV RNA” for the isolation of EV RNA.6.1.1.1.3 Precipitation of EVs from adherent cells

[0250] For adherent cells grown in culture media containing exosome-depleted FBS for 1-3 days and are 70%-90% confluent at the time of EV harvest, the following steps are performed for precipitation of EVs from adherent cells:

[0251] Cells grown in 6-well tissue culture plates are ideal for EV harvest.

[0252] Collect 1.4 mL of conditioned cell culture media in a microcentrifuge tube. Do not let the pipet tip touch the bottom of the plate.

[0253] As an optional step, save the plate containing attached cells for harvesting cellular RNA as directed in section 3b.

[0254] As a negative control collect 1.4 mL of culture media containing exosome- depleted FBS (no cells) in a microcentrifuge tube.

[0255] Centrifuge the samples at 3,000 x g for 5 minutes in a microcentrifuge.

[0256] Transfer 1.2 mL of supernatant into a fresh microcentrifuge tube being careful not to transfer pelleted material.

[0257] Centrifuge the 1.2 mL samples at 12k RPM for 15 minutes.

[0258] Transfer 1.0 mL of supernatant to a fresh microcentrifuge tube being careful not to transfer pelleted material. Store samples on ice.

[0259] Add 200 pL of ExoQuick TC Tissue Culture Media Exosome Precipitation Solution (SBI) to each sample. (Note: ExoQuick-TC is viscous and difficult to pipet. When working with this reagent pipet slowly using wide-bore pipet tips. Ensure that the entire 200 pL volume is added to each sample).

[0260] Vortex samples well. Visually inspect the samples to ensure that they are properly mixed.

[0261] Store samples at 4°C for at least 18 hours to precipitate EVs.

[0262] Precipitated EV samples can be stored for up to 2 weeks at 4°C.

[0263] Proceed to section “isolation of EV RNA”.6.1.1.1.4 Isolation of cellular RNA from suspension cells

[0264] Cellular RNA is 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. Each sample requires 100 mL of SingleShot cell lysis master mix. The steps for isolating the cellular RNA from suspension cells with the SingleShot cell lysis kit is provided below:

[0265] Prepare SingleShot cell lysis master mix for the appropriate number of samples plus 10% (Table 5). Store on ice.

[0266] Table 1. Preparation of SingleShot Cell Lysis Master Mix:

[0267] Starting material is the residual cells from step 6 in section 2a.

[0268] Add 1 mL of PBS to each residual cell sample. Vortex well.

[0269] Remove 100 mL of cell sample into a fresh microcentrifuge tube.

[0270] Centrifuge samples at 3,000 x g for 5 minutes in a microcentrifuge. Discard supernatant.

[0271] Pulse spin the samples and remove all traces of residual liquid.

[0272] Add 100 mL of SingleShot cell lysis master mix to each sample. Resuspend the precipitated EVs by vortexing.

[0273] Transfer the samples into 0.2 mL PCR strip tubes. Store on ice.

[0274] Incubate samples in a PTC Tempo deep well thermocycler (or equivalent) according to the protocol listed in Table 2.Table 2. Thermal Cycling Protocol. Set heated lid temperature to 105°C.

[0275] Store EV RNA samples at -80°C.

[0276] Proceed to section “Fragmentation of cellular RNA” for the fragmentation of cellular RNA.6.1.1.1.5 Isolation of cellular RNA from adherent cells

[0277] Cellular RNA is 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. Each sample requires 200 mL of SingleShot cell lysis master mix. The steps for isolating the cellular RNA from adherent cells with the SingleShot cell lysis kit is provided below:

[0278] Prepare SingleShot cell lysis master mix for the appropriate number of samples plus 10% (Table 3). Store on ice.

[0279] Table 3. Preparation of SingleShot Cell Lysis Master Mix for harvesting cellular RNA from adherent cells

[0280] Starting material are the attached cells remaining in the well of the 6-well tissue culture plate from step 3 in section “Precipitation of EVs from adherent cells”.

[0281] Add 1 mL PBS to each well containing cells to be analyzed. Mix gently. Aspirate liquid completely.

[0282] Re-wash the cells with 1 mL of PBS. Aspirate liquid completely.

[0283] Add 200 mL of SingleShot cell lysis master mix to each well. Gently tilt and rotate the plate to evenly distribute the SingleShot mastermix over the cells. Let incubate at room temperature for 5 minutes.

[0284] Transfer the SingleShot cell lysis mix into 0.2 mL PCR strip tubes. Store on ice.

[0285] Incubate samples in a PTC Tempo deep well thermocycler or (equivalent) according to the protocol listed in Table 2 in section “Isolation of cellular RNA from suspension cells”.

[0286] Store EV RNA samples at -80°C.

[0287] Proceed to section 5b for the fragmentation of cellular RNA6.1.1.1.6 Isolation of EV RNA

[0288] EV RNA samples are isolated from EV for RNA-seq using the SingleShotCell Lysis Kit (Bio-Rad). Each sample requires 100 pL of SingleShot cell lysis master mix. To isolate RNA from EVs, RNA samples from EVs were diluted 10-fold in SingleShot buffer containing DNAse and proteinase K. 100 pL of SingleShot buffer containing DNase and proteinase K was used to resuspend the isolated EV pellet. The EVs were then incubated at 37°C for 5 minutes followed by a 75°C incubation for 5 minutes. The samples were then cooled to 4°C and stored at -80°C.

[0289] Prepare SingleShot cell lysis master mix for the appropriate number of samples as directed in Table 1 of “Isolation of cellular RNA from suspension cells”. Store on ice.

[0290] Centrifuge the precipitated EV samples at 12k RPM for 15 minutes. Discard supernatant.

[0291] Pulse spin the samples and remove all traces of residual liquid.

[0292] Add 100 pL of SingleShot cell lysis master mix to each sample. Resuspend the precipitated EVs by vortexing. Store on ice.

[0293] Incubate samples according to the directions in table 6 of section 3a.

[0294] Store EV RNA samples at -80°C.

[0295] Proceed to section “preparation of EV RNA”6.1.1.2 Part 2. Preparation of RNA-Seq libraries6.1.1.2.1 Preparation of EV RNA

[0296] This section describes preparation of the RNA-Seq libraries of EV RNA. The steps are provided below:

[0297] Thaw SEQuoia Complete reagents A-E and RNA samples completely before use. Store on ice.

[0298] Prepare an RNA fragmentation master mix for the appropriate number of samples plus 10% (Table 4). Store on ice.

[0299] Table 4. Preparation of RNA fragmentation Master Mix

[0300] In 0.2 mL PCR tubes, aliquot 13pL of RNA fragmentation master mix. Store on ice.

[0301] Add 5 pL of EV RNA sample to each tube.

[0302] Mix tubes by vortexing gently, then centrifuge to collect reactions at the bottom of the tubes.

[0303] Store tubes on ice.

[0304] Proceed to section “ end repair”.

[0305] Note that EV RNA samples are not fragmented prior to RNA-Seq library preparation, whereas cellular RNA are fragmented. If analyzing both EV and cellular RNA samples, ensure that they are in separate 0.2 mL PCR strip tubes.6.1.1.2.2 Fragmentation of cellular RNA

[0306] This section describes preparation of the RNA-Seq libraries of cellular RNA. The steps are provided below:

[0307] Thaw SEQuoia Complete reagents A-E and RNA samples completely before use.

[0308] Preheat thermal cycler to 94°C with a heated lid temperature of 105°C.

[0309] Prepare an RNA fragmentation mastermix according to the directions in Table 4, section “Preparation of EV RNA”.

[0310] In 0.2 mL PCR tubes, aliquot 13 pL of RNA fragmentation mastermix. Store on ice.

[0311] Add 5 pL of cellular RNA sample to each tube.

[0312] Mix tubes by vortexing gently, then centrifuge to collect reactions at the bottom of the tubes.

[0313] Incubate tubes in a PTC Tempo deep well thermocycler (or equivalent) according to the protocol in Table 5.Table 5. Thermal Cycling Protocol. Set heated lid temperature to 105°C.

[0314] Store tubes on ice.

[0315] Proceed to section “end repair”.6.1.1.2.3 End repair

[0316] The protocol for “End repair” is provided in the steps below:

[0317] Preheat thermal cycler to 37°C with a heated lid temperature of 105°C.

[0318] Add 2 pL of Reagent B, End Repair Enzyme to each sample.

[0319] Mix reactions by vortexing. Pulse centrifuge to collect samples at the bottom of the tubes.

[0320] Incubate tubes in a PTC Tempo deep well thermocycler (or equivalent) according to the protocol in Table 6.Table 6. Thermal Cycling Protocol. Set heated lid temperature to 105°C.

[0321] Place reactions on ice for 5 minutes.

[0322] Proceed to section “Poly(A) tailing”.6.1.1.2.4 Poly(A) tailing

[0323] The protocol for Poly (A) tailing is provided in the steps below:

[0324] Preheat thermal cycler to 16°C with a heated lid temperature of 105°C.

[0325] Prepare Poly(A) tailing master mix for the appropriate number of samples plus10% (Table 7). Store on ice.Table 7. Preparation of Poly(A) Tailing Master Mix

[0326] Add 25 pL of poly(A) master mix to each reaction.

[0327] Mix reactions by vortexing. Pulse centrifuge to collect samples at the bottom of the tubes.

[0328] Incubate in a PTC Tempo deep well thermocycler (or equivalent) according to the protocol in Table 8.Table 8. Thermal Cycling Protocol. Set heated lid temperature to 105°C.

[0329] Place reactions on ice for 5 minutes.

[0330] Proceed with section 8, continuous synthesis.6.1.1.2.5 Continuous synthesis

[0331] Preheat thermal cycler to 34°C with a heated lid temperature of 105°C.

[0332] Add 5 pL of Reagent E, SEQzyme mix to each reaction.

[0333] Mix reactions by vortexing. Pulse centrifuge to collect samples at the bottom of the tubes.

[0334] Incubate in a PTC Tempo deep well thermocycler (or equivalent) according to the protocol in Table 9.Table 9. Thermal Cycling Protocol. Set heated lid temperature to 105°C.

[0335] Place reactions on ice for 5 minutes

[0336] This is a safe stopping point. Samples can be stored at 4°C overnight or at - 20°C if storing for longer than 24 hours.

[0337] When ready, proceed to section “cDNA purification”.6.1.1.2.6 cDNA purification

[0338] Allow Purification Beads to come to room temperature.

[0339] Vortex beads to resuspend.

[0340] Add 20 pL of purification beads to each sample.

[0341] Mix well by pipetting up and down several times.

[0342] Incubate at room temperature for 20 minutes.

[0343] Place the tubes on a magnetic rack for 5-10 minutes or until solution is clear.

[0344] Transfer the supernatant, containing cDNA, to a fresh 0.2 mL PCR tube.

[0345] Proceed to section “cDNA concentration”.6.1.1.2.7 cDNA concentration

[0346] 1. Bring SPRIselect Reagent (Beckman Coulter) to room temperature.

[0347] 2. Add 112 pL of SPRIselect Reagent to the supernatant collected at the end of the cDNA purification step (section 9, step 7).

[0348] 3. Mix well by pipetting up and down several times.

[0349] 4. Incubate at room temperature for 5-8 minutes.

[0350] 5. Centrifuge reactions briefly to collect the reactions at the bottom to the tubes.

[0351] 6. Place the tubes on a magnetic rack for 5 minutes or until the solution clears.

[0352] Note: Keep tubes on magnetic rack through step “Allow beads to dry at room temperature for 2-4 minutes. Do not over dry the beads”.

[0353] 7. Aspirate and discard the cleared solution.

[0354] 8. Wash beads by adding 200 pL of freshly prepared 80% ethanol. Incubate for 30 seconds.

[0355] 9. Aspirate and discard the ethanol without disturbing the beads.

[0356] 10. Repeat the ethanol wash step (steps 8 and 9) removing all traces of ethanol.

[0357] 11. Allow beads to dry at room temperature for 2-4 minutes. Do not over dry the beads.

[0358] 12. Remove tubes from the magnetic rack.

[0359] 13. Add 24 pL of IDTE, pH 8.0 to each tube and mix well by pipetting up and down several times.

[0360] 14. Incubate the tubes at room temperature for 2 minutes.

[0361] 15. Centrifuge the reactions briefly and place the tubes on a magnetic rack for5 mins or until the solution clears.

[0362] 16. Transfer 22.5pL of the supernatant containing cDNA to a fresh PCR 0.2 mL tube.

[0363] This is a safe stopping point. Samples can be stored at 4°C overnight or at - 20°C if storing for longer than 24 hours.

[0364] 18. When ready, proceed to section “determination of the number of library amplification PCR cycles”.6.1.1.2.8 Determination of the number of library amplification qPCR cycles

[0365] For samples processed using qPCR, the present inventors found that EV RNA is significantly different than cellular RNA and requires a real-time PCR step to determine the optimal number of library amplification PCR cycles. Cellular RNA samples also benefit from this step. The steps are provided below:

[0366] SYBR green I nucleic acid gel stain (ThermoFisher) is at a high concentration in DMSO. Thaw at room temperature and dilute to 2X, in DMSO.

[0367] Thaw RNA samples and no cell control sample(s) on ice. Thaw amplification mix (green cap) from the SEQuoia Complete kit and a separate SEQuoia Dual Indexed Primer, one for each sample, on ice.

[0368] Prepare library PCR master mix for the appropriate number of samples plus 10% (Table 10). Store on ice.Table 10. Preparation of Library PCR Master Mix

[0369] Aliquot 36.5 pL of library PCR master mix into individual wells of a 96-well PCR plate. Include a well for the no cell control sample.

[0370] Add 11 pL of sample from section “cDNA concentration”, the step of “Transfer 22.5pL of the supernatant containing cDNA to a fresh PCR 0.2 mL tube into each well”.

[0371] Carefully pierce the wells of the 96-well dual index primer plate and add 2.5 pL of SEQuoia dual index primer to each well of the PCR plate. Make sure that each well gets a different index primer.

[0372] Seal the plate with optical sealing film. Vortex the plate and centrifuge for 2 minutes at 4000 rpm to collect the reactions at the bottom of the plate and eliminate bubbles.

[0373] A CFX Opus 96 real-time PCR instrument (Bio-Rad) running CFX Maestro software version 2.0 (or later) was used to perform this experiment. Other real-time PCR instruments can be used.

[0374] 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 that signal detection in the SYBR channel is specified.Table 11. Thermal Cycling Protocol

[0375] In order to determine the number of amplification cycles for the libraries, the data was analyzed using 2 different thresholds which effectively set lower and upper boundaries for the cycles during which exponential amplification is taking place. This is described in the following section:

[0376] Following the RT-PCR run, open the run file using CFX Maestro software.

[0377] Make a rough estimate of the maximal RFU of the amplification traces. For the example amplification plot shown in FIG. 2A 30,000 RFU was used as the maximal value (red, horizontal line) even though some amplification traces have a maximal value higher of lower than 30,000 RFU.

[0378] Set the baseline threshold to 25% of the maximal RFU and collect Cq values in whole numbers.

[0379] Next, Cq values were collected in CFX Maestro program.

[0380] Set the baseline threshold to 75% of the maximal RFU and collect Cq values in whole numbers. FIG. 2B shows an amplification plot with baseline thresholds set at 25% and 75% of the maximal RFU (purple, horizontal lines). Table 12 shows the whole number Cq value of each sample at 25% and 75% of the maximal RFU.Table 12. Cq values at 25% and 75% of the maximal RFU

[0381] The number of library amplification PCR cycles to perform is anywhere between and including the Cq values at 25% and 75% max RFU. The number of amplification cycles can vary between individual samples. For simplicity, samples are grouped such that the number of library amplification runs are minimized. In the example shown in table 13 three library amplification experiments should be performed with 10, 13 and 16 amplification cycles. Samples 9 and 11 can be run for either 10 or 13 amplification cycles. Sample 10 can be run for either 13 or 16 amplification cycles.Table 13. Number of library amplification cycles to perform for each sample

[0382] Proceed to section “RNA-Seq library amplification and indexing”.6.1.1.2.9 RNA-Seq library amplification and indexing

[0383] 1.Using data collected in section “Determination of the number of library amplification PCR cycles” determined how many separate library amplification PCR runs will need to be performed. For the example experiment shown in Table 13 three library amplification PCR runs will need to be performed (10, 13 and 16 cycles).

[0384] 2.Collect the appropriate RNA samples for one library amplification PCR run and thaw on ice.

[0385] 3.Thaw amplification mix (green cap) from the SEQuoia Complete kit and the same SEQuoia Dual Indexed Primers used on the samples in section 11. Store on ice.

[0386] 4. Prepare RNA-Seq library amplification master mix for the appropriate number of samples plus 10% (Table 14). Store on ice.Table 14. Preparation of RNA-Seq Library Amplification Master Mix

[0387] 5. Aliquot 36.5 pL of library PCR master mix into 0.2 mL strip tubes.

[0388] 6.Add 11 pL of sample from section 10, step 16 into each tube.

[0389] 7. Add 2.5 pL of SEQuoia dual index primer to each tube. Make sure that each sample gets the same dual index primer it received in section “Determination of the number of library amplification PCR cycles”.

[0390] 8. Amplify the samples on a PTC Tempo deep well thermocycler (or equivalent) according to the protocol listed in Table 15.Table 15. RNA-Seq Library Amplification Protocol

[0391] Repeat the library amplification PCR run, steps 2-8, with a different number of PCR cycles (if necessary).

[0392] Proceed to section “post amplification clean-up”.6.1.1.2.10 Post-amplification clean-up

[0393] 1.Bring SPRIselect Reagent (Beckman Coulter) to room temperature.

[0394] 2 Add 60 pL of SPRIselect Reagent to each tube.

[0395] 3. Mix well by pipetting up and down several times.

[0396] 4. Incubate at room temperature for 5-8 mins.

[0397] 5. Centrifuge reactions briefly to collect the reactions at the bottom to the tubes.

[0398] 6. Place the tubes on a magnetic rack for 5 mins or until the solution clears.

[0399] Note: Keep tubes on magnetic rack through step 11.

[0400] 7. Aspirate and discard the cleared solution.

[0401] 8. Wash beads by adding 200 pL of freshly prepared 80% ethanol. Incubate for 30 secs.

[0402] 9. Aspirate and discard the ethanol without disturbing the beads.

[0403] 10. Repeat the ethanol wash step (steps 8 and 9) removing all traces of ethanol.

[0404] 11. Allow beads to dry at room temperature for 2-4 mins. Do not over dry the beads.

[0405] 12. Remove tubes from the magnetic rack.

[0406] 13. Add 20 pL of IDTE, pH 8.0 to each tube and mix well by pipetting up and down several times.

[0407] 14. Incubate the tubes at room temperature for 2 minutes.

[0408] 15. Centrifuge the reactions briefly and place the tubes on a magnetic rack for5 mins or until solution clears.

[0409] 16. Transfer 18 pL of the supernatant containing the RNA-Seq libraries to a fresh PCR tube.

[0410] 17. This is a safe stopping point. Samples can be stored at 4°C overnight or at-20°C if storing for longer than 24 hours.

[0411] 18. Proceed to section “assessment of the quality and quantity of RNA-Seq libraries”.6.1.1.2.11 Assessment of library quality and quantity

[0412] The quality and quantity of RNA-Seq libraries are determined using an Agilent 2100 Bioanalyzer or equivalent using High Sensitivity DNA Chips, according to manufacturer instructions. Example Bioanalyzer traces analyzing a cellular RNA- Seq library or an EV RNA-Seq library are shown in FIG. 3 A and 3B respectively.6.1.1.3 Assays and Primers

[0413] Table 16 lists primers and Table 17 lists assays including combinations of primers."+" indicates that the following base is a locked nucleic acid (LNA)6.1.1.4 Part 4: Processing of RNA-Seq Data6.1.1.4.1 Seq Sense Data Analysis Pipeline

[0414] There are 3 steps to processing and analyzing SEQuoia Complete Stranded RNA data:

[0415] 1. FASTQ preprocessing: Trimming of SEQuoia complete specific Poly(A) tails

[0416] 2. Alignment: Single-pass alignment to a conjoined annotation set of the transcriptome

[0417] 3 Feature counting - Output of mapped reads with raw and normalized counts(TPM, RPKM)

[0418] The FASTQ files generated on the instrument or on Illumina Basespace were downloaded and a Web-based Seq-Sense Platform (https: / / seqsense.bio-rad.com / ) was used to analyze the RNA Sequencing data.

[0419] The Seq-Sense platform was launched and the SEQuoia Complete as the kit was selected as the kit of interest.

[0420] The FASTQ files were uploaded and acquired from the instrument or Basespace server.

[0421] The pipeline parameters were then set and processed for the experiment. In this study, hg38 was used as species and enabled UMI processing to perform deduplication of reads (both R1 and R2 files were present). The recommended read length suggested are 75 bp for R1 and 8 bp of R2. The first 8 bases of R2 consists of a random tag sequence that can be used as a unique molecular identifier (UMI).6.1.2. Part 5: Analysis of RNA-Seq Data

[0422] RNA-Seq FASTQ data was processed using the SeqSense NGS Data Analysis Software (Bio-Rad). RNA transcript structure was determined by converting the output BAM files to bigWig files (bin size = 1) and visualizing the bigWig files as custom tracks on the UCSC genome browser.6.1.2.1 Analysis of target gene reads

[0423] The output bam files from the sequencing were uploaded onto the Galaxy Server (https: / / usegalaxy.org / ).

[0424] In the sub-menu, select bam as the file type and Human 2013 (hg38) as the genome of interest.

[0425] Next, the bam reads were converted to the bigwig format that is easy to visualize on the UCSC genome browser.

[0426] The tool parameters were set to bin size in bases as 1 and hg38 as the genome of interest.

[0427] The bigwig files were visualized on UCSC genome browser as shown in FIG. 4. Under custom tracks, the configuration was set to “full”. The tracks auto scale to show the gene expression levels.

[0428] Record gene expression pattern for gene of interest.6.1.2.2 Analysis of transcript structure6.1.2.2.1 Design of PCR assays

[0429] Transcript fragmentation assessment PCR assay sets were designed, based on RNA-Seq data, targeting transcripts that are intact in cellular RNA and fragmented in EV RNA. RNA transcript structure was determined by visualizing cellular RNA and EV RNA expression using bigWig tracks on the UCSC genome browser. Each assay set consists of two assay types, a control assay and a test assay. Control assays amplify RNA regions that are expressed in both cellular and EV RNA. Test assays amplify RNA regions that are expressed in cellular RNA, but less so in EV RNA due to it being fragmented (FIGs. 1A-1L). Each PCR assay set may have more than one control or test assay. If more than one control or test assay is used to evaluate a target, the Cq values of the PCR reactions are averaged. Six PCR assay sets were developed. The oligonucleotide sequences for each primer are listed in table 16 and the assays are listed in table 17.6.1.2.2.2 PCR analysis of cellular and EV RNA

[0430] RNA isolated from cells and EVs for PCR analysis were 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. Cq values of samples analyzed without reverse transcriptase reflect the level of background DNA. In all samples the level of contaminating DNA was negligible and did not impact the PCR results (Table 3). qPCR Method: qPCR Reaction Steps

[0431] When using qPCR, assessment of RNA structure was performed using ACq values comparing the control and test assays. Assays analyzing EV RNA are expected to have a large negative ACq value whereas assays analyzing cellular RNA are expected to have a relatively smaller ACq value that could be negative or positive.

[0432]

[0433] The steps for qPCR using the iTaq Universal SYBR Green One-Step Kit is provided below:

[0434] Collect the appropriate RNA samples for qPCR validation of RNA samples and thaw on ice.

[0435] Thaw iTaq Univ SYBR Green 1-Step and i Script Reverse Transcriptase, and 4x assay. Store on ice.

[0436] Prepare qPCR master mix for the appropriate number of samples plus 10% (Table 18). Store on ice.Table 18. Preparation of qPCR validation Master Mix

[0437] Aliquot 20.25 pL of library PCR master mix into individual wells of a 96-well PCR plate. Include a well for the no cell control sample.

[0438] Seal the plate with optical sealing film. Vortex the plate and centrifuge to collect the reactions at the bottom of the plate. qPCR Method: qPCR Thermal Cycling

[0439] When using qPCR, 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 19.Table 19. Thermal Cycling Protocol

[0440] After the first 50°C step produces cDNA, the protocol immediately proceeds with PCR amplification and monitoring fluorescence. Following the RT-PCR run, open the run file using CFX Maestro software and collect the Cq values from the “Quantification tab”.

[0441] Assays analyzing EV RNA are expected to have a large negative DCq value.

[0442] Assays analyzing cellular RNA are expected to have a relatively smaller DCq value that could be negative or positive.6.1.3. qPCR Results6.1.3.1 EV transcripts are fragmented

[0443] The data showed striking differences when comparing RNA-Seq results analyzing RNA isolated from EV particles with results analyzing cellular RNA. It was observed that many EV transcripts were fragmented when compared with their intact cellular counterparts. In addition, the pattern of EV transcript fragmentation was generally consistent in different cell lines (see FIGs. 5A-5L).

[0444] Several patterns of EV transcript fragmentation were observed. One pattern, exemplified in FIG. 5A-5L, consisted of separate RNA pieces at the 5’ and 3’ ends of the transcript with few transcripts containing bases in the middle. Another pattern,exemplified by FIG. 5J and FIG. 5K, showed an EV transcript that encompassed the middle portion of the gene with few bases as the 5’ and 3’ ends. The EVs SCARNA5 transcript consisted of two regions in the middle of the gene and few bases at the 5’ and 3’ ends, a combination of the two transcript fragmentation patterns described previously (see FIG. 5L).6.1.3.2 PCR assays that differentiate EV and cellular RNA based on transcript structure

[0445] The fragmented transcript structure of EV RNAs observed by RNA-Seq was intriguing. To provide additional proof that these structures exist PCR technology was utilized to develop a new type of assay, the “Transcript Fragmentation Assessment PCR Assay”. The assay strategy involves designing a control PCR assay that amplifies an RNA region that is present in both EV and cellular RNA, and a test PCR assay that should, based on the RNA-Seq data, amplify cellular RNA, but not amplify EV RNA because the amplicon includes an RNA region that is mostly absent in EVs due to transcript fragmentation. RNA transcript structure is determined from the ACq value comparing the control and test assays. Assays analyzing EV RNA are expected to have a large ACq because the control assay should amplify EV RNA and have a Cq value whereas the test PCR assay should not amplify EV RNA well and have a Cq value significantly delayed from the control. When cellular RNA is analyzed with these assays the ACq value is expected to be relatively small because both control and test assays should amplify intact transcripts with a similar Cq. Thus, these assay sets assess transcript structure with large negative ACq values reflecting fragmented transcripts and small ACq values implying that the transcripts are intact. AACq values, which compare the EV and cellular ACq values, quantifies the difference in target gene transcript structure between the cellular compartment and the EV compartment.

[0446] Designing PCR assays that target the fragmented transcripts found in EVs can be difficult because many fragments are small (under 30 bases) and unable to accommodate forward and reverse PCR primers. In some instances, we incorporated locked nucleic acid bases into the PCR primers to increase the primer Tm and improve PCR performance. Despite the difficulties in designing PCR primers, transcript fragmentation assessment PCR assay sets were designed for six of the targets shown in FIGs. 5A-5L. The PCR assay designs and results are presented in FIGs. 6A-6D (RNY5), FIGs. 7A-7D (SCARNA10), FIGs. 8A-8D (SCARN5A), FIGs. 9A-9D (SCARNA10), FIGs. 10A-10D (RMRP), and FIGs. 11A-11D (RNY1).

[0447] Transcript fragmentation assessment PCR assay sets targeting RNY5 (FIGs. 6A-6D), RNU12 (FIGs. 7A-7D), SCARNA5 (FIGs. 8A-8D), SCARNA10 (FIGs. 9A-9D) and RMRP (FIGs. 10A-10D) confirm the fragmented transcript structure of these target genes in EVs observed by RNA-Seq (FIGs. 6A, 7A, 8A, 9A, and 10A, respectively). The AACq values range from -5.40 to -14.69 in the different cell lines, indicating that the fragmented transcript structure is at least 42-fold more prevalent in EV RNA relative to cellular RNA.

[0448] The RNY1 transcript has separate 5’ and 3’ fragments in the EVs of all cell lines analyzed (FIGs. 11A-11D). However, the AACq values range from -1.93 to - 4.83 which place the RNY1 EV transcript structure closer to cellular RNA than the other targets quantified by the transcript fragmentation assessment PCR (AACq range -5.40 to -14.69; see RNY5 (FIGs. 6A-6D), RNU12 (FIGs. 7A-7D), SCARNA5 (FIGs. 8A-8D), SCARNA10 (FIGs. 9A-9D) and RMRP (FIGs. 10A-10D)). Visual inspection of the RNY1 RNA-Seq read profile suggests that the transcript is incompletely fragmented, which may reflect the less negative AACq scores (see FIG. HA)6.1.4. Discussion

[0449] This data provides evidence that EV transcripts are fragmented. Previous work analyzing Y-RNAs supports this theory. Y-RNAs are small, non-coding RNAs that interact with the RO60 and SSB proteins to form a ribonucleoprotein (RNP) complex that is functionally implicated in DNA replication, RNA quality control and the cellular stress response (Valkov, N., Advances in Experimental Medicine and Biology, Springer, 2020, pp. 327-342. doi: 10.1007 / 978-981-15-1671-9_20). There are four human Y-RNAs that are encoded by the RNY1, RNY3, RNY4 and RNY5 genes. The RNY5 transcript structure in EVs isolated from human BJ and K562 cells was previously determined by Northern blot analysis and found to have the same split 5’ and 3’ structure observed in our RNA-Seq results (Chakrabortty, S.K., RNA, 21(11): 1966-1979 (2015)) (FIGs. 12A-12B). This provides further evidence, from Northern technology, that EV transcripts are fragmented in human samples. In addition, the location of the transcript fragments is consistent with the locations observed in our RNA-Seq experiments providing additional support for the existence of a fragmented RNY5 EV transcript structure (see FIGs. 12A-12B).

[0450] Previous work analyzing Y-RNA / RO60 interaction by X-ray crystallography provides insights as to how transcript fragmentation occurs in some instances, whichhelps to interpret the data in the current study. Y-RNA bases that are critical for RO60 protein interaction were mapped to an 8-base sequence at the 5’ end and a 7-base sequence at 3’ end of the Y-RNA transcript (Stein, A.J. Cell, 121(4): 529-539, May 2005) (FIG. 13A). The 5’ sequence motif is conserved in all human RNY genes and starts at the 4thbase from the 5’ end of the transcripts. The first six bases of the RO60 3’ binding site are also conserved in all human RNY genes. However, the final base of the 3’ motif is not conserved in the human RNY4 gene where it is a G rather than an A (FIG. 13B-13E). Interestingly, all RO60 interaction sites are found within a transcript region that is highly expressed in EVs. This suggests the possibility that RO60 bound to RNY transcripts protects the bound regions from nuclease digestion. RMRP, which is abundant in EVs (see FIGs. 5E, 10A-10D), is the RNA component of a RNP complex that has ribonuclease activity (Hermanns, P., Hum Mol Genet, 14(23): 3723-3740 (2005)). It is plausible that ribonuclease activity in EVs can digest unprotected RNA and fragment RNY transcripts into the pieces observed by RNA- Seq.

[0451] It is perhaps significant that all of the fragmented EV transcripts identified in this study are small non-coding RNAs that are components of functional RNP complexes. It is possible that the fragmented transcript pieces found in EVs are there because they are bound by protein and protected from digestion by ribonucleases. The distinction between free floating RNA and RNA that is a component of a functional RNP complex is an important factor to consider in understanding the EV transcriptome.

[0452] The RNY genes are found in abundance in human blood (Dhahbi, J.M., Physiol Genomics, 45: 990-998 (2013), doi: 10.1152 / physiolgenomics.00129.2013) and are being studied for use as biomarkers that monitor human health (C. Gulia et al., “Cancers, 12(5) (2020) doi: 10.3390 / cancersl2051238.) and Driedonks et al., J Extracell Vesicles, 9(1) (2020), doi: 10.1080 / 20013078.2020.1764213). The transcript fragmentation assessment PCR assays that assess RNY1 and RNY5 may be useful tools to follow these markers.6.2. Example 2: Transcript Fragmentation Assessment ddPCR assay

[0453] This experiment assesses the primer designs and test / control assays used in Example 1 in a droplet digital PCR assay to analyze and distinguish EV RNAs from cellular RNAs based on transcript structure differences.

[0454] As shown in FIG. 1C, EV RNA and cellular RNA samples were cultured, precipitated, isolated, and lysed as described in Example 1. After lysing the EV RNA and cellular RNA samples, cDNA is prepared from EV and cellular SingleShot lysates.6.2.1.1 Preparation of cDNA

[0455] iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad #1725035) was used to prepare cDNA from EV lysates (14ul undiluted lysate into reaction) and cellular lysates (diluted 1 :50 in TE) in bulk using iScript gDNA Clear cDNA Synthesis kit. This was a 2 step process: gDNA clearance followed by cDNA synthesis.

[0456] The steps for preparing cDNA are provided below:

[0457] Prepare DNase mater mix:Table 20: DNA Master Mix

[0458] Add 2ul mix to 14ul sample (if less than 14ul make up to 14 w / H20

[0459] After mixing, place in thermal cycler:Table 21: DNase reaction protocol in thermocycler

[0460] Add 4 ul RT Supermix to each 16ul reaction, place in thermal cycler:Table 22: cDNA synthesis reaction protocol in thermocycler

[0461] Transfer supernatant containing cDNA to a fresh PCR tube.6.2.1.2 ddPCR reaction setup

[0462] After cDNA synthesis, ddPCR reactions were set up using ddPCR EvaGreen Supermix. EV cDNA was used directly (5ul) in 20ul ddPCR reactions (QX200 ddPCR EvaGreen Supermix, Bio-rad #186-4034) containing 250nM primers. The EvaGreen Supermix was used according to manufacturer’s instructions. QX200 ddPCR EvaGreen Supermix is a 2x concentrated, ready-t-use reaction cocktail containing all components, except primers and template, required for droplet digital PCR. 250 nM primers were added to the reaction.

[0463] Cellular cDNA was diluted 1 :2 in water prior to using 5ul per 20ul EvaGreen reaction with 250nM primers.

[0464] The Preparation of the reaction mix is provided below:

[0465] Table 23: Preparation of the Reaction Mix:

[0466] Table 24: Master Mix Preparation:6.2.1.3 Droplet generation

[0467] Droplets were generated with an Automated Droplet Generator (1864101) with QX200 Droplet Generation Oil for EvaGreen (Bio-Rad #1864006). About 20,000 droplets are generated from a 20 pL reaction. Droplets are formed using a specific oil with the droplet generator. Once the droplets are formed, they were placed on a 96 well plate.

[0468] Droplet counts were averaged for 2 replicate wells. If more than one control or test assay was employed, the droplet counts were averaged.6.2.1.4 Thermal Cycling

[0469] Thermal cycling was carried out in PTC Tempo Deepwell Thermal Cycler (Bio-Rad #12015392) according to reagent instructions and manufacturer instructions. The cDNA of the EV RNA and cellular RNA samples were amplify ed while the droplets were hardened and stabilized.6.2.1.5 Droplet Reading

[0470] Droplets were counted on a QX200 Droplet Digital PCR System QX200 (or any Bio-Rad droplet reader, including QX600). Data was analyzed using QX Manager software.

[0471] Droplets were read using reader oil. All of the contents of the well were run through the reader.

[0472] The QX software identified a threshold when there are both positive and negative droplets. For example, if there are only positive droplets, the cellular RNA template needs to be diluted further further and repeated.6.2.1.6 Data Analysis

[0473] As described in “design of PCR assays” of Example 1, transcript fragmentation assessment PCR assay sets were designed, based on RNA-Seq data, targeting transcripts that are intact in cellular RNA and fragmented in EV RNA. RNA transcript structure was determined by visualizing cellular RNA and EV RNA expression using bigWig tracks on the UCSC genome browser. Each assay set consists of two assay types, a control assay and a test assay. Control assays amplify RNA regions that are expressed in both cellular and EV RNA compartments. Test assays amplify RNA regions that are expressed in cellular RNA, but less so in EV RNA due to it being fragmented. Each PCR assay set may have more than one control or test assay.

[0474] As shown in FIGs. 14-19, Transcript fragmentation using ddPCR is determined by first calculating a “Delta Copy number”, 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 calculating a first ratio (X) of “Delta Copy number” and “Average Test” for a cellular RNA sample (e.g., first compartment x(ceiiuiar RNA) ) and a EV RNA sample (e.g., second compartment X(EV RNA)), and a second ratio of x(ceiiuiar RNA) to X(EV RNA).. A first amplified product includes the reverse complement of a first RNA sequence of an RNA transcript that is at most partially fragmented (e.g., “test” assay containing RNA regions that are expressed in both cellular and EV RNA compartments) and a second amplified product includes the reverse complement of a fragmented portion of the first RNA sequence (e.g., “control”).

[0475] For RMRP as shown in FIG. 14 used in ddPCR, the ratio of the second amplified product and the first amplified product in the A549 cell line show that the RMRP transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0476] For RNY5 as shown in FIG. 15 used in ddPCR, the ratio of the second amplified product and the first amplified product in the A549 cell line show that the RNY5 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0477] For RNU12 as shown in FIG. 16 used in ddPCR, the ratio of the second amplified product and the first amplified product in the A549 cell line show that theRNU12 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0478] For SCARNA5, as shown in FIG. 17 used in ddPCR, the ratio of the second amplified product and the first amplified product in the A549 cell line show that the SCARNA5 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0479] For SCARNA10, as shown in FIG. 18 used in ddPCR, the ratio of the second amplified product and the first amplified product in the A549 cell line show that the SCARNA10 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results.

[0480] For RNY1, as shown in FIG. 19 used in ddPCRthe ratio of the second amplified product and the first amplified product in the A549 cell line show that the RNY 1 transcript is highly fragmented in EV RNA relative to cellular RNA, which is similar to qPCR results. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0481] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting fragmentation of an RNA transcript in a sample, the method comprising: adding to the sample a first primer pair and a second primer pair, wherein the sample comprises: a first RNA sequence of the RNA transcript that is at most partially fragmented; a second RNA sequence of the RNA transcript, wherein all or a portion 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 to and amplifying all or a portion of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the first RNA fragment; amplifying the first RNA sequence and the first RNA fragment using the first and second primer pairs, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence and a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment; quantifying the amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on the amount of the second amplified product, wherein the amount of the second amplified product indicates RNA transcript fragmentation.

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

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

4. A method of detecting fragmentation of an RNA transcript in a sample, the method comprising: adding to the sample at least a first primer pair, a second primer pair, and a third primer pair, wherein the sample comprises: a first RNA sequence of the RNA transcript that is at most partially fragmented; and a second RNA sequence of the RNA transcript, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and an optional a second RNA fragment, the first primer pair is capable of hybridizing to and amplifying all or a portion of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the first RNA fragment; and the third primer pair is capable of hybridizing to and amplifying the second RNA fragment; amplifying the first RNA sequence, the first RNA fragment, and the second RNA fragment using the first, second, and third primer pairs, respectively, thereby generating a first amplified product comprising the sequence or reverse complement thereof of the first RNA sequence, a second amplified product comprising the sequence or reverse complement thereof of the first RNA fragment, and a third amplified product comprising the sequence or reverse complement thereof of the second RNA fragment; quantifying: the amount of the first amplified product, the amount of the second amplified product; and the amount of the third amplified product; and detecting fragmentation of the RNA transcript in the sample based on the average amount of the second amplified product and the third amplified product, wherein the average amount of the second amplified product and the third amplified product indicates RNA transcript fragmentation.

5. The method of claim 4, wherein detecting comprises determining the amount of fragmentation of the RNA transcript in the sample relative to the amount of the first amplified product is based on the average amount of the second amplified product andthe third amplified product, wherein a comparison between the average amount of the second amplified product and the third amplified product relative to the first amplified product indicates the amount of RNA transcript fragmentation.

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

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

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

11. The method of 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 second compartment by determining the amount or degree of the first RNA sequence and first or second RNA fragment of the second RNA sequence in a first compartment and / or second compartment.

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

13. The method of claim 8-12, further comprising one or more additional compartments.

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

15. The method of any one of claims 8-14, wherein the first compartment comprises RNA originating from a plurality of cells (e.g., cellular compartment), a plurality of extracellular vesicles (EVs) / exosomes (e.g., EV compartment), nuclear RNA (e.g., nuclear compartment), cytoplasmic RNA (e.g., cytoplasmic compartment), mitochondrial RNA (e.g., mitochondrial compartment), chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

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

17. The method of any one of claims 8-16, wherein: the first compartment, the second compartment, or both, comprise RNA originating from a control sample; or the first compartment comprises RNA originating from a control sample comprising a plurality of cells, and a second compartment comprises RNA originating from a test sample suspected to be fragmented, wherein the RNA originating from the test sample is selected from: a plurality of extracellular vesicles / exosomes, nuclear RNA, cytoplasmic RNA, mitochondrial RNA, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

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

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

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

21. The method of any one of claims 8-20, wherein: the first RNA sequence, the second RNA sequence, or both are derived from the RNA transcript; and wherein 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 deemed 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 the RNA transcript is deemed to be expressed in both the first compartment comprising plurality of cells and the second compartment comprising the plurality of extracellular vesicles based in part on RNA sequencing data from the plurality of cells and RNA-sequencing data from the plurality of extracellular vesicles / exosomes.

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

27. The method of any one of claims 25-26, wherein the amount of the first amplified product from the first compartment comprising the plurality of cells is more than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

28. The method of any one of claims 24-27, wherein the first RNA fragment and second RNA fragment in the second RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles.

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

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

31. The method of any one of claims 22-30, wherein the second primer pair is capable of hybridizing to 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 of any one of claims 22-26, wherein a third primer pair is capable of hybridizing to 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 of any one of claims 23-32, wherein a 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 as a reduction in the number of sequencing reads mapping to the nucleotides in the second RNA sequence separating the first RNA fragment and the second RNA fragment as compared to the number sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.

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

35. The method of claim 34, wherein the non-coding RNA is selected from a Y-RNA, a snRNA, a ncRNA, a snoRNA, a snaRNA, a tRNA, a rRNA, a scRNA, a telomerase RNA, a vault RNA, a guide RNA, a miRNA, an antisense RNA, piRNA, and a IncRNA.

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

37. The method of claim 36, wherein the RNA-sequencing data of the first RNA sequence from the first compartment shows the first RNA sequence is at most partially fragmented.

38. The method of 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 shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment.

40. The method of 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 of 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, selected 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 shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment.

43. The method of 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 of 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 of 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 of any one of claims 1-45, wherein fragmenting of the second RNA sequence is not the result of sonication, tagmentation, chemical modification, UV irradiation, or heat degradation.

47. The method of any one of claims 1-46, wherein fragmentation of the second RNA sequence is due at least in part to the presence of exonucleases, endonucleases, RNA processing enzymes, an RNA binding protein bound to the second RNA sequence, or a combination thereof.

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

49. The method of any one of claims 1-48, wherein the second amplified product, the third amplified 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 amplified product, the third amplified 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 of any one of claims 1-49, wherein the second amplified product, the third amplified product, or both range from 20 to 80 nucleotides in length.

51. The method of any one of claims 1-45, wherein before said amplifying, 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 transcribing the first RNA sequence and the first RNA fragment into complementary DNA (cDNA).

53. The method of any one of claims 1-52, wherein the first primer pair comprises:(i) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4;(ii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7;(iii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8;(iv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10;(v) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12;(vi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15;(vii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or(viii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22.

54. The method of any one of claims 1-53, wherein the second primer pair comprises(i) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2;(ii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4;(iii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8;(iv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10;(v) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14;(vi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17;(vii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or(viii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

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

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

57. A method for designing primers for detecting fragmentation of an RNA transcript, the method comprising: analyzingRNA-sequencing data of RNA collected from a first compartment, and RNA sequencing data of RNA collected from a second compartment, whereinthe RNA-sequencing data from the first compartment comprises a first RNA sequence that is at most partially fragmented; the RNA sequencing data from the second compartment comprises the first RNA sequence, wherein all or a portion 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 that is capable of hybridizing to and amplifying the first RNA sequence, and a second primer pair that is capable of hybridizing to the first RNA fragment.

58. A method for designing primers for detecting fragmentation of an RNA transcript, the method comprising: analyzingRNA-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 comprises a first RNA sequence that is at most partially fragmented; the RNA sequencing data from the second compartment comprises the first RNA sequence, wherein all or a portion 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 that is capable of hybridizing to and amplifying the first RNA sequence; a second primer pair that is capable of hybridizing to and amplifying the first RNA fragment; anda third primer pair that is capable of hybridizing to and amplifying the second RNA fragment.

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

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

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

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

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

64. A method of detecting fragmentation of an RNA transcript in a sample, the method comprising: reverse transcribing the RNA transcript into complementary DNA (cDNA); adding to the sample a first primer pair and a second primer pair, wherein: the sample comprises: a reverse complement of a first RNA sequence of the RNA transcript that is at most partially fragmented to produce a reverse complement of the first RNA sequence; a reverse complement of a second RNA sequence of the RNA transcript in the sample, wherein all or a portion 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 to and amplifying all or a portion of the reverse complement of the first RNA sequence; the second primer pair is capable of hybridizing to and amplifying the reverse complement of the first RNA fragment;amplifying the reverse compliment of the first RNA sequence and the reverse complement of the first RNA fragment using the first and second primer pairs, thereby generating a first amplified product comprising the reverse complement of the first RNA sequence and a second amplified product comprising the reverse complement of the first RNA fragment; quantifying the amount of the first amplified product and the amount of the second amplified product; and detecting fragmentation of the RNA transcript in the sample based on a ratio of the amount of the second amplified product and the amount of the first amplified product.

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

66. The method of any one of claims 64-65, wherein after adding to the sample the first primer pair and the second primer pair but before said amplifying, the method comprises partitioning the sample into a plurality of droplets.

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

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

69. The method of any one of claims 66-67, wherein the dilution ranges from 1 :2 to 1 :50.

70. The method of any one of claims 66-69, further comprising: adding to the sample a third primer pair, wherein the third primer pair is capable of hybridizing to and amplifying the second RNA fragment; amplifying the second RNA fragment using the third primer pair in a separate reaction from the first RNA sequence and the first RNA fragment, thereby generating a third amplified product comprising the sequence or reverse complement thereof of the second RNA fragment; quantifying: the amount of the third amplified product; and detecting fragmentation of the RNA transcript in the sample based on one or more of:a ratio of the first amplified product, the second amplified product, and the third amplified product; a ratio of the first amplified product and the second amplified product; a ratio of the first amplified product and the third amplified product; a ratio of the first amplified product and the average amount of the second amplified product and the third amplified product; a ratio of the second amplified product and the first amplified product; a ratio of the second amplified product, the third amplified product, and the first amplified product; a ratio of the third amplified product and the first amplified product; and a ratio of the average amount of the second amplified product and the third amplified product, and the first amplified product.

71. The method of any one of claims 65-70, wherein the sample is partitioned into the plurality of droplets comprising: a first set of droplets, each droplet comprising the reverse complement of the first RNA sequence (cDNA), the first primer pair, and one or more reagents; a second set of droplets, each droplet comprising the reverse complement of the first RNA fragment, the second primer pair, and one or more reagents; optionally a third set of droplets, each droplet comprising the reverse complement of the second RNA fragment, a third primer pair, and one or more reagents; and / or a subset of droplets that do not comprise the reverse complement of the first RNA sequence, the reverse complement of the first RNA fragment, and / or the reverse complement of the second RNA fragment.

72. The method of any one of claims 70-71, wherein the method further comprises: adjusting the second amplicon product and / or the third amplicon product 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); anddetecting fragmentation of the RNA transcript in the sample is based on one or more of: a ratio of the first amplified product originating from a plurality of cells, the adjusted second amplified product originating from a plurality of cells, and the adjusted third amplified product originating from the plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes, the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes, and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the adjusted second amplified product originating from a plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of cells; a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the first amplified product originating from a plurality of cells and the average amount of the adjusted second amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of cells; and a ratio of the first amplified product originating from a plurality of extracellular vesicles / exosomes and the average amount of the adjusted second amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the adjusted second amplified product originating from a plurality of cells, the adjusted third amplified product originating from a plurality of cells, and the first amplified product originating from the plurality of cells; a ratio of the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes, the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes, and the first amplified product originating from a plurality of extracellular vesicles / exosomes;a ratio of the adjusted second amplified product originating from a plurality of cells and the first amplified product originating from a plurality of cells; a ratio of the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the adjusted third amplified product originating from a plurality of cells and the first amplified product originating from a plurality of cells; a ratio of the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes and the first amplified product originating from a plurality of extracellular vesicles / exosomes; a ratio of the average amount of the adjusted second amplified product originating from a plurality of cells and the adjusted third amplified product originating from a plurality of cells, and the first amplified product originating from a plurality of cells; and a ratio of the average amount of the adjusted second amplified product originating from a plurality of extracellular vesicles / exosomes and the adjusted third amplified product originating from a plurality of extracellular vesicles / exosomes, and the first amplified product originating from a plurality of extracellular vesicles / exosomes.

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

74. The method of 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 gap 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 of 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 gap 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, atleast 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 of any one of claims 64-75, wherein the sample comprises RNA from a first compartment, a second compartment, or both.

77. The method of 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 second compartment.

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

79. The method of claim 76-78, further comprising one or more additional compartments.

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

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

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

83. The method of any one of claims 76-82, wherein the first compartment, the second compartment, or both, comprise RNA originating 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, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

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

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

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

87. The method of any one of claims 64-86, wherein the first RNA sequence, the second RNA sequence, or both are: derived from the RNA transcript, or are 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 deemed 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, chloroplast, vacuoles, lysosomes, endoplasmic reticulum, or a combination thereof.

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

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

93. The method of any one of claims 91-92, wherein the amount of the first amplified product from the first compartment comprising the plurality of cells is more than the amount of the first amplified product from the second compartment comprising the plurality of extracellular vesicles.

94. The method of any one of claims 91-93, wherein the first RNA fragment and second RNA fragment in the second RNA sequence is expressed in the first compartment comprising the plurality of cells and the second compartment comprising the plurality of extracellular vesicles.

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

96. The method of any one of claims 88-95, wherein the first primer pair is capable of hybridizing to 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 of any one of claims 88-96, wherein the second primer pair is capable of hybridizing to 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 for the first and second compartments is determined using the following formulas:Delta Copy Numb epfirst compartment) = the amount of the second amplified product - the amount of the first amplified product;Delta Copy Numbepsecond compartment) = the amount of the second amplified product - the amount of the first amplified product;X(first compartment)=Delta Copy Number(first compartment) originating from the plurality of cells amount of first amplified product in compartment originating from the plurality of cells ’ andX(second compartment)=Delta Copy Number(second compartment) originating from the plurality of extracellular vescicles / exosomes amount of first amplified product in compartment originating from the plurality of extracellular vescicles / exosomes99. The method of claim 98, detecting fragmentation of the RNA transcript in the sample based is further based on a second ratio, wherein the second ratio is determined based( 1n . , < . Xffirst compartment) on the following formula: second ratio = — - - - or second ratio =X(secona compartment)X(second compartment) X(first compartment)100. The method of claim 99, wherein the second ratio is not 1.

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

102. The method of any one of claims 88-101, wherein a 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 as a reduction in the number of sequencing reads mapping to the nucleotides in the second RNA sequence separating the first RNA fragment and the second RNA fragment as compared to the number sequencing reads mapping to the corresponding nucleotides in the first RNA sequence in the first compartment.

103. The method of any one of claims 64-103, wherein the RNA transcript is a noncoding RNA.

104. The method of claim 103, wherein the non-coding RNA is selected from a Y- RNA, a snRNA, a ncRNA, a snoRNA, a snaRNA, a tRNA, a rRNA, a scRNA, a telomerase RNA, a vault RNA, a guide RNA, a miRNA, an antisense RNA, piRNA, and a IncRNA.

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

106. The method of claim 105, wherein the RNA-sequencing data of the first RNA sequence from the first compartment shows the first RNA sequence is at most partially fragmented.

107. The method of 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 shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment.

109. The method of 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 selected 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 shows the second RNA sequence is fragmented into the first RNA fragment and second RNA fragment.

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

115. The method of any one of claims 64-114, wherein fragmenting of the second RNA sequence is not the result of sonication, tagmentation, chemical modification, UV irradiation, or heat degradation.

116. The method of any one of claims 64-115, wherein fragmentation of the second RNA sequence is due at least in part to the presence of exonucleases, endonucleases, RNA processing enzymes, an RNA binding protein bound to the second RNA sequence, or a combination thereof.

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

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

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

120. The method of any one of claims 64-119, wherein the first primer pair comprises:(ix) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4;(x) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7;(xi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8;(xii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10;(xiii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12;(xiv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15;(xv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or(xvi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22.

121. The method of any one of claims 64-120, wherein the second primer pair comprises:(ix) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2;(x) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4;(xi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8;(xii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10;(xiii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14;(xiv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17;(xv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or(xvi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

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

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

124. A set of primers, comprising: a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence, wherein the first RNA sequence is at most partially fragmented; and a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

125. A set of primers, comprising: a first primer pair that is capable of hybridizing to and amplifying a first RNA sequence wherein the first RNA sequence is at most partially fragmented;a second primer pair that is capable of hybridizing to and amplifying a first RNA fragment of a second RNA sequence; and a third primer pair that is capable of hybridizing to and amplifying a second RNA fragment of a second RNA sequence, wherein all or a portion of the second RNA sequence is fragmented into at least a first RNA fragment and a second RNA fragment.

126. A set of primers, comprising: a first primer pair comprising(i) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4;(ii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 5 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 7;(iii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8;(iv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10;(v) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 11 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 12;(vi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 15;(vii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17 and a reverse primer having asequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or(viii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 22; a second primer pair comprising(i) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 2;(ii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 3 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 4;(iii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 6 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 8;(iv) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 9 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 10;(v) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 13 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 14;(vi) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 16 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 17;(vii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 18 and a reverse primer having asequence with at least 80% sequence identity to a sequence of SEQ ID NO: 19; or(viii) a forward primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 20 and a reverse primer having a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 21.

127. The set of primers of any one of claims 124-126, wherein the primers in the first primer pair, the primers in the second primer pair, both comprise one or more modified nucleotides.

128. The set of primers of claim 127, wherein the modified nucleotide is a locked nucleotide.

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

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

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