Method for detecting target miRNA and its precursor

Abstract

To provide a method for detecting miRNAs and their precursors contained in trace amounts in samples, such as serum, with a high degree of accuracy and making it possible to distinguish between diseases by comparing the detected amounts between samples.SOLUTION: Provided is a method for detecting target miRNAs and their precursors in a sample. The method comprises: an RNA extraction step of extracting RNA in the sample; an adapter ligation step of ligating an adapter to the 5' end and / or 3' end of the extracted RNA; a reverse transcription step of obtaining a reverse transcription product by reverse transcription of the adapter-ligated RNA obtained in the adapter ligation step; an amplification step of obtaining an amplification product by amplifying the reverse transcription product by a nucleic acid amplification method; obtaining a nucleic acid molecule containing a desired length range from the amplification product; and a base sequencing step of determining the base sequence of the nucleic acid molecule obtained in the obtaining step. The desired length is 15 to 33 bases in length excluding the adapter portion.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting target miRNAs and their precursors, and a method for assisting in the diagnosis of disease. [Background technology]

[0002] PCR, microarray, mass spectrometry, etc. have been used to detect nucleic acids in samples such as cells and tissues to analyze individual disease information. In addition to these, methods for detecting nucleic acids using next-generation sequencing have been attracting attention in recent years.

[0003] Next-generation sequencing is a technology that makes it possible to simultaneously obtain sequence information for a huge number of DNA fragments. This technology has dramatically increased the amount of sequence information that can be obtained in a single analysis compared to conventional technologies such as PCR and microarray. It is expected that applying next-generation sequencing to the analysis of nucleic acids in biological samples obtained from subjects will enable the analysis of disease information with extremely high accuracy.

[0004] When analyzing a subject's disease information from nucleic acids in a biological sample, RNAs such as messenger RNA (mRNA) and microRNA (miRNA) are the primary targets of analysis. However, ribosomal RNA (rRNA) generally accounts for the majority of all RNA, at approximately 80%, followed by transfer RNA (tRNA) at over 10%. Meanwhile, mRNA accounts for only a few percent, and miRNA accounts for an even smaller proportion. Therefore, when analyzing miRNA, a step is required to remove noise components such as rRNA from the sample before sequencing. However, miRNA sequences are short, at 20–25 base pairs, making them difficult to separate from other RNAs.

[0005] For example, Illumina's method (Non-Patent Document 1) proposes a library preparation method in which a 5' adapter and a 3' adapter are ligated to both ends of the miRNA, followed by reverse transcription and PCR amplification, followed by electrophoresis, followed by excision from the gel of a 145-160 bp range encompassing the combined length of the mature miRNA (21-25 bases) and the adapter sequence (130 bases, including the 5' and 3' ends). In such cases, it is common knowledge among those skilled in the art to exclude nucleic acid fragments derived from rRNA, etc., from the target range in order to reduce noise. In the above example, it is necessary to avoid the inclusion of nucleic acid fragments exceeding 160 bp in size. While this method may result in a reduced recovery rate of miRNA, it is effective when targeting cells, tissues, etc. that contain relatively high amounts of miRNA.

[0006] On the other hand, when targeting samples containing very small amounts of miRNA, such as serum, the recovery rate of miRNA from the sample is extremely important, and the impact of miRNA loss during removal of impurities using gels or magnetic beads cannot be ignored. Therefore, it is not possible to obtain sufficient amounts of miRNA from samples such as serum to detect differences in expression levels, and it has not been possible to distinguish diseases such as cancer based on the detection results.

[0007] Therefore, a new technique is needed to detect differences in expression levels of miRNAs between specimens, even when the specimens contain very small amounts of miRNA. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 8,999,677 [Non-patent literature]

[0009] [Non-Patent Document 1] Illumina, Inc., "Small RNA Expression Analysis Achieved with BaseSpace," March 28, 2016 Summary of the Invention [Problem to be solved by the invention]

[0010] The object of the present invention is to provide a method for detecting miRNAs and their precursors contained in trace amounts in samples such as serum with high accuracy, and for distinguishing between diseases by comparing the detected amounts between samples. [Means for solving the problem]

[0011] To solve the above-mentioned problems, the present inventors obtained nucleic acid molecules from the serum of cancer patients and healthy individuals, contrary to conventional technical knowledge, so as to contain not only mature miRNAs but also miRNA precursors, and then performed sequencing using next-generation sequencing. As a result, when comparing the expression levels of either mature miRNAs or precursor miRNAs alone, no difference was detected between cancer specimens and healthy specimens. However, the present inventors found that a difference between cancer specimens and healthy specimens could only be detected when the combined expression levels of mature miRNAs and precursor miRNAs were used as the comparison control, leading to the completion of the present invention. The present invention is based on this finding and provides the following:

[0012] (1) A method for detecting a target miRNA and its precursor in a sample, comprising: an RNA extraction step of extracting RNA in the sample; an adapter ligation step of ligating an adapter to the 5' end and / or 3' end of the extracted RNA; a reverse transcription step of reverse transcribing the adapter-ligated RNA obtained in the adapter ligation step to obtain a reverse transcription product; an amplification step of amplifying the reverse transcription product by a nucleic acid amplification method to obtain an amplified product; and an amplification step of amplifying the amplified product to obtain a target miRNA having a length in a desired range. Base length range The method includes an acquisition step of acquiring a nucleic acid molecule, and a base sequence determination step of determining the base sequence of the nucleic acid molecule acquired in the acquisition step, and the length of the target range is 15 to 33 bases excluding the adaptor portion. and the base length range of the nucleic acid molecule obtained in the obtaining step is such that the lower limit Lmin of the base length range excluding the adaptor portion is 15 or less, and the upper limit Lmax of the base length range excluding the adaptor portion is 100 or more. method. (2) The upper limit Lmax of the base length range is 200 or more. , The method described in (1). (3) The method according to (1) or (2), wherein the base sequence is determined using next-generation sequencing. (4) A method for detecting a target miRNA and its precursor in a sample, comprising: an RNA extraction step of extracting RNA from the sample; and extracting RNA having a length in a desired range from the extracted RNA. Base length range The method includes an acquisition step of acquiring a nucleic acid molecule, and a base sequence determination step of determining the base sequence of the nucleic acid molecule acquired in the acquisition step, wherein the length of the target range is 15 to 33 bases long. and the base length range of the nucleic acid molecule obtained in the obtaining step is such that the lower limit Lmin of the base length range excluding the adaptor portion is 15 or less, and the upper limit Lmax of the base length range excluding the adaptor portion is 100 or more. method. (5) The method according to (4), wherein the nucleic acid molecule obtained in the obtaining step is 100 bases or less in length. (6) The method according to (4) or (5), wherein the base sequence is determined using direct RNA sequencing. (7) The method according to (3) or (6), further comprising a measuring step of measuring the number of reads of the target miRNA, its precursor, and / or its fragment. (8) The method described in (7), wherein the target miRNA is one or more selected from the group consisting of hsa-miR-16-5p, hsa-miR-483-3p, hsa-miR-4492, hsa-miR-3648-1, and hsa-miR-663a. (9) The method according to any one of (1) to (8), wherein the sample is a body fluid.

[0013] (10) A method for assisting in the diagnosis of a disease, comprising: an RNA extraction step of extracting RNA from body fluids derived from a subject and a control; an adapter ligation step of ligating an adapter to the 5' end and / or the 3' end of the extracted RNA; a reverse transcription step of reverse transcribing the adapter-ligated RNA obtained in the adapter ligation step to obtain a reverse transcription product; an amplification step of amplifying the reverse transcription product by a nucleic acid amplification method to obtain an amplified product; and a method for detecting a target length range from the amplified product. Base length rangeThe method includes an acquisition step of acquiring a nucleic acid molecule, a base sequence determination step of determining the base sequence of the nucleic acid molecule acquired in the acquisition step by next-generation sequencing, a step of measuring the number of reads of the target miRNA, its precursor, and / or its fragment, and a step of comparing the number of reads of the subject and a control and determining that the subject is suffering from a disease if the number of reads of the subject is different from the number of reads of the control, and the length of the target range is 15 to 33 bases excluding the adaptor portion. and the base length range of the nucleic acid molecule obtained in the obtaining step is such that the lower limit Lmin of the base length range excluding the adaptor portion is 15 or less, and the upper limit Lmax of the base length range excluding the adaptor portion is 100 or more. method. (11) A method for assisting in the diagnosis of a disease, comprising: an RNA extraction step of extracting RNA from body fluids derived from a subject and a control; an acquisition step of acquiring nucleic acid molecules having a length within a desired range from the extracted RNA; a base sequence determination step of determining the base sequence of the nucleic acid molecule acquired in the acquisition step by direct RNA sequencing; a step of measuring the number of reads of a target miRNA, its precursor, and / or its fragment; and a step of comparing the number of reads of the subject and the control, and determining that the subject has a disease if the number of reads of the subject is different from the number of reads of the control, wherein the length within the desired range is 15 to 33 bases excluding the adaptor portion. and the base length range of the nucleic acid molecule obtained in the obtaining step is such that the lower limit Lmin of the base length range excluding the adaptor portion is 15 or less, and the upper limit Lmax of the base length range excluding the adaptor portion is 100 or more. method. (12) The method according to (10) or (11), wherein the control subject is a healthy subject. (13) The method according to any one of (10) to (12), wherein the body fluid is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, tissue fluid, and saliva. (14) The method according to any one of (10) to (13), wherein the disease is cancer. (15) The method according to (14), wherein the cancer is breast cancer or pancreatic cancer. (16) The method according to (14), wherein the cancer is breast cancer and the target miRNA is one or more selected from hsa-miR-16-5p, hsa-miR-483-3p, and hsa-miR-4492. (17) The method according to (14), wherein the cancer is pancreatic cancer and the target miRNA is hsa-miR-3648-1 or hsa-miR-663a. [Effects of the Invention]

[0014] The present invention provides a method for detecting with high accuracy miRNAs contained in trace amounts in samples such as serum, and for distinguishing between diseases by comparing the amount of detected miRNA between samples. [Brief explanation of the drawings]

[0015] [Figure 1]

[0023] Figure 1 illustrates an example of the range of nucleic acid molecules obtained in the obtaining step of the method of the present invention. In the obtaining step, nucleic acid molecules are obtained so that the range of base lengths, excluding the adaptor portion, is 15 to 33 bases. Part of the range of nucleic acid molecules obtained in the obtaining step is illustrated by the double arrow in the figure. [Figure 2] Figure 1 shows the number of reads of mature miRNAs, precursor miRNAs, and both mature and precursor miRNAs detected in the serum of breast cancer patients and healthy individuals. The figure shows the number of reads of mature miRNAs, precursor miRNAs, and both mature and precursor miRNAs of hsa-miR-16-5p. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1. Method for detecting target miRNA and its precursor (when reverse transcription step is included) 1-1. Overview The first aspect of the present invention is a method for detecting a target miRNA and its precursor. The detection method of this aspect includes, as essential steps, an RNA extraction step, an adapter ligation step, a reverse transcription step, an amplification step, an acquisition step, and a base sequencing step, and is capable of detecting a target miRNA and its precursor with high accuracy from samples containing very small amounts of miRNA, such as serum. The detection method of this aspect differs from the detection method of the second aspect in that it includes a reverse transcription step and subjects the nucleic acid after the reverse transcription step to base sequencing.

[0017] 1-2.Definition of Terms The abbreviations used in this specification, such as "nucleotide," "polynucleotide," "DNA," and "RNA," shall be in accordance with the "Guidelines for the Preparation of Specifications Including Nucleotide Sequences or Amino Acid Sequences" (edited by the Japan Patent Office) and common usage in the art.

[0018] As used herein, the term "polynucleotide" refers to nucleic acids, including RNA, DNA, and RNA / DNA chimeras. The term "DNA" includes cDNA, genomic DNA, and synthetic DNA. The term "RNA" includes total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. As used herein, "synthetic DNA" and "synthetic RNA" refer to DNA and RNA artificially produced using, for example, an automated nucleic acid synthesizer, based on a predetermined base sequence (which may be either a natural or non-natural sequence). As used herein, the term "non-natural sequence" is intended to be used in a broad sense and includes sequences that differ from a natural sequence, such as sequences containing one or more nucleotide substitutions, deletions, insertions, and / or additions (i.e., mutant sequences), sequences containing one or more modified nucleotides (i.e., modified sequences), and the like. As used herein, the term "polynucleotide" is used interchangeably with the term "nucleic acid."

[0019] As used herein, the term "fragment" refers to a polynucleotide having a continuous partial nucleotide sequence of a polynucleotide. Herein, it particularly refers to a fragment of miRNA or a miRNA precursor. Fragments of miRNA or miRNA precursors are preferably long enough to identify the miRNA gene from the read nucleotide sequence, and desirably have a length of, for example, 12 or more bases, 15 or more bases, preferably 17 or more bases, and more preferably 19 or more bases.

[0020] As used herein, the term "gene" is intended to encompass not only RNA and double-stranded DNA, but also each of the single-stranded DNAs that constitute them, such as the positive strand (or sense strand) or complementary strand (or antisense strand), and is not particularly limited by its length.

[0021] Therefore, unless otherwise specified, the term "gene" as used herein includes double-stranded DNA including human genomic DNA, single-stranded DNA (positive strand), single-stranded DNA (complementary strand) having a sequence complementary to the positive strand (e.g., cDNA), microRNA (miRNA), fragments thereof, and transcription products thereof. Note that the term "gene" does not limit the functional region and can include, for example, an expression control region, a coding region, an exon, or an intron.

[0022] As used herein, the term "transcription product" refers to RNA synthesized using the DNA sequence of a gene as a template. RNA polymerase binds to a site called the promoter located upstream of the gene, and RNA is synthesized by attaching ribonucleotides to the 3' end so that the sequence is complementary to the DNA base sequence. This RNA includes not only the gene itself, but also the entire sequence from the transcription start point to the end of the poly(A) sequence, including expression control regions, coding regions, exons, and introns.

[0023] As used herein, a "primer" refers to an oligonucleotide or polynucleotide that specifically recognizes a portion of a polynucleotide chain and serves as the starting point for a polymerase reaction using the polynucleotide chain as a template. A primer is usually single-stranded and preferably has a base sequence complementary to a portion of the polynucleotide chain that serves as a template for the polymerase reaction.

[0024] As used herein, an "adapter" refers to an oligonucleotide or polynucleotide that is linked to the 5' and / or 3' ends of a polynucleotide chain. Herein, the adapters linked to the 5' and 3' ends of a polynucleotide chain are referred to as 5' and 3' adapters, respectively. In the present invention, the adapter may be either a DNA adapter or an RNA adapter, but is preferably an RNA adapter.

[0025] Unless otherwise specified, the term "microRNA (miRNA)" as used herein refers to a 15-25 or 19-25 base non-coding RNA that is transcribed as a hairpin-like RNA precursor, cleaved by a dsRNA cleaving enzyme with RNase III cleavage activity, incorporated into a protein complex called RISC, and involved in mRNA translational repression. miRNAs are encoded by miRNA genes on the genome, and there are more than 1,000 types of miRNA genes in the human genome. miRNA genes are transcribed into single-stranded RNA by RNA polymerase II, which then binds within the RNA sequence to form a hairpin loop-shaped double-stranded structure. miRNAs with this structure are called primary miRNAs (pri-miRNAs). The pri-miRNA is cleaved by the Drosha protein to form precursor miRNAs (pre-miRNAs), which are then processed by the Dicer protein to form double-stranded miRNAs. One strand of the double-stranded miRNA binds to an Argonaute protein (herein referred to as "Ago protein") to form an RNA-induced silencing complex (RISC). As used herein, miRNA refers to the mature miRNA unless otherwise specified.

[0026] As used herein, the term "miRNA precursor" refers to an immature miRNA that can be generated during the process of miRNA maturation. Specific examples of miRNA precursors include, but are not limited to, the aforementioned pri-miRNA and pre-miRNA.

[0027] The term "hsa-miR-16-5p" as used herein includes hsa-miR-16-5p (miRBase Accession No. MIMAT0000069) set forth in SEQ ID NO: 1, as well as homologs or orthologs from other species. Furthermore, a known precursor of hsa-miR-16-5p is hsa-mir-16-1 (miRBase Accession No. MI0000070, SEQ ID NO: 6), which has a hairpin-like structure.

[0028] The term "hsa-miR-483-3p" as used herein includes hsa-miR-483-3p (miRBase Accession No. MIMAT0002137) set forth in SEQ ID NO: 2, as well as homologs or orthologs from other species. Furthermore, a known precursor of hsa-miR-483 is hsa-mir-483 (miRBase Accession No. MI0002467, SEQ ID NO: 7), which has a hairpin-like structure.

[0029] The term "hsa-miR-4492" as used herein includes hsa-miR-4492 (miRBase Accession No. MIMAT0019027) set forth in SEQ ID NO: 3, as well as homologs or orthologs from other species. Furthermore, a known precursor of hsa-miR-4492 is hsa-mir-4492 (miRBase Accession No. MI0016854, SEQ ID NO: 8), which has a hairpin-like structure.

[0030] The term "hsa-miR-3648-1" as used herein includes hsa-miR-3648-1 (miRBase Accession No. MIMAT0018068) set forth in SEQ ID NO: 4, as well as homologs or orthologs from other species. Furthermore, a known precursor of hsa-miR-3648-1 is hsa-mir-3648-1 (miRBase Accession No. MI0016048, SEQ ID NO: 9), which has a hairpin-like structure.

[0031] The term "hsa-miR-663a" as used herein includes hsa-miR-663a (miRBase Accession No. MIMAT0003326) set forth in SEQ ID NO: 5, as well as homologs or orthologs from other species. Furthermore, a known precursor of hsa-miR-663a is hsa-mir-663a (miRBase Accession No. MI0003672, SEQ ID NO: 10), which has a hairpin-like structure.

[0032] "Next-generation sequencing (NGS)" is a collective term used to distinguish recently developed sequencing methods from the first-generation Sanger sequencing (dideoxygenation). NGS encompasses second-generation sequencing methods and subsequent developments, such as third-generation sequencing. Second-generation sequencing methods simultaneously determine the base sequences of enormous numbers of DNA fragments, ranging from tens to hundreds of millions. Specific examples include pyrosequencing, sequencing by synthesis, ligation sequencing, and ion-conducting sequencing. Pyrosequencing is a method based on the detection of pyrophosphate released during DNA synthesis, which is detected using luciferase enzymes. Synthetic sequencing uses reversible terminators to determine the sequence of individual bases. Ligation sequencing is a method based on the mismatch sensitivity of DNA ligase. Ion semiconductor sequencing is a method based on the detection of hydrogen ions released during DNA synthesis. Examples of third-generation sequencing methods include single-molecule sequencing methods such as single-molecule real-time sequencing. In this specification, all second-generation and later sequencing methods are considered to be included in next-generation sequencing. A DNA fragment analyzed in next-generation sequencing is called a "read (read fragment)," and the number of times a DNA fragment is read is called the "number of reads." The length of the read (base length) is called the "read length."

[0033] "Single-molecule sequencing" is a sequencing method that enables the acquisition of sequence data with longer reads than second-generation next-generation sequencing. Specific examples of single-molecule sequencing include methods using PacBio RSII and Sequel (Pacific Biosciences) as single-molecule sequencers, and methods using nanopore sequencers such as MinION (Oxford Nanopore Technology).

[0034] "Direct RNA sequencing" refers to a method for determining the sequence of an RNA molecule itself without reverse transcription. Conventional methods for sequencing RNA generally require converting RNA to cDNA, whereas direct RNA sequencing can determine the RNA sequence without the bias of reverse transcription or PCR. An example of a direct RNA sequencing method is nanopore sequencing, which uses a nanopore sequencer such as MinION (Oxford Nanopore Technology).

[0035] "Nanopore sequencing" is a method in which an adapter sequence with a motor protein attached is added to the end of the nucleic acid molecule to be sequenced, and the nucleic acid molecule is pushed by the motor protein into a nanoscale hole called a nanopore, and the base sequence is determined by the change in current that occurs when the nucleic acid molecule passes through the membrane. Nanopore sequencing can be performed using MinION, PromethION, GridION, Flongle, SmidgION, etc., provided by Oxford Nanopore Technology.

[0036] The term "detection" as used herein may be replaced with the terms "test," "measurement," "determination," or "determination support." Furthermore, the term "evaluation" as used herein is used to mean supporting a diagnosis or evaluation based on test results or measurement results.

[0037] As used herein, the term "subject" refers to mammals such as humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, horses, sheep and goats, rodents such as mice and rats, and animals kept in zoos. A preferred subject is a human.

[0038] As used herein, a "sample" to be detected or diagnosed refers to a sample collected from a subject, a healthy subject, or a group of healthy subjects and subjected to the detection method of the present invention. Examples of such samples include body fluids, cells, tissues, and other fluids such as feces and hair. Examples of body fluids include cerebrospinal fluid, interstitial fluid, blood (including serum, plasma, and interstitial fluid), lymph, tissue or cell extracts, pleural effusion, sputum, tears, nasal discharge, saliva, and urine. Blood may be serum or plasma prepared from blood. Tissues and cells include, for example, tissues and cells of a subject that are affected or potentially affected by a disease, as well as corresponding tissues and cells in a healthy subject, such as tissues or cells derived from the liver, pancreas, lung, esophagus, kidney, ovary, stomach, colon, prostate, or breast. In addition to the above, samples may also include biological samples extracted from these, specifically RNA such as miRNA.

[0039] As used herein, the term "healthy individual" refers to an individual not suffering from a specific disease, preferably an individual not suffering from any disease. However, as used herein, healthy cells are also included in the broad definition of healthy individuals. Therefore, a healthy individual refers not only to an individual level, but also to a cellular level, such as a normal portion of tissue collected from a cancer patient, if the healthy state is present.

[0040] As used herein, "P" or "P value" indicates the probability that a statistical value that is more extreme than the statistical value actually calculated from data under the null hypothesis will be observed in a statistical test. Therefore, the smaller the "P" or "P value," the more significant the difference between the compared subjects.

[0041] As used herein, "several" means an integer of about 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0042] In this specification, "plurality" refers to, for example, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0043] 1-3. Method The detection method of this embodiment includes, as essential steps, an RNA extraction step, an adapter ligation step, a reverse transcription step, an amplification step, an acquisition step, and a base sequencing step, and includes, as a selection step, a measurement step.

[0044] (RNA extraction process) The "RNA extraction step" is a step of extracting RNA from a sample. In one embodiment, the sample may be a bodily fluid. Preferred examples of bodily fluids include blood, serum, plasma, cerebrospinal fluid, urine, interstitial fluid, and saliva.

[0045] The method for extracting RNA from a sample is not particularly limited. For example, the common acid phenol method (Acid Guanidinium-Phenol-Chloroform (AGPC) method) may be used, or an RNA extraction reagent containing acid phenol may be used. Examples of RNA extraction reagents that can be used in this step include the RNA extraction reagent in the 3D-Gene® RNA extraction reagent from liquid sample kit (Toray Industries, Inc.), Trizol® (Life Technologies), and Isogen (Nippon Gene Co., Ltd., Japan). Furthermore, kits such as the miRNeasy® Mini Kit (Qiagen) can also be used, but the method is not limited to these. RNA extraction kits are commercially available from life science manufacturers such as Qiagen, Takara Bio, Toyobo, Thermo Fisher Scientific, and Promega, and these kits can also be used.

[0046] When RNA is extracted from tissue as a sample, it is preferable to dissolve the tissue in a solution containing a surfactant and / or protease before RNA extraction in order to increase the RNA yield, but when using body fluids such as serum, the treatment with a surfactant and / or protease may be omitted. Furthermore, the RNA after RNA extraction can be purified as necessary.

[0047] (Adapter connection process) The "adapter ligation step" is a step of ligating an adapter to the 5' end and / or 3' end of the RNA extracted in the RNA extraction step.

[0048] The 5' adapter ligated to the 5' end and the 3' adapter ligated to the 3' end can be ligated to RNA in either order, or simultaneously. Typically, the 3' adapter is ligated first, followed by the 5' adapter, to ligate the 5' and 3' adapters to the 5' and 3' ends, respectively. For example, the 5' end of the RNA can be dephosphorylated, followed by ligating the 3' adapter to the 3' end of the RNA, and then phosphorylating the 5' end of the RNA, followed by ligating the 5' adapter to the 5' end of the RNA. While RNA ligases such as T4 RNA ligase can be used in such ligation reactions, the dephosphorylation / phosphorylation steps can be omitted by using an RNA ligase mutant capable of selectively ligating an adapter to the 3' end of RNA, such as T4 RNA ligase 2, truncated.

[0049] The 5' adapter preferably has a sequence complementary to the nucleotide sequence of the primer so that the primer can bind thereto in the amplification step described below, and the 3' adapter preferably has a sequence complementary to the nucleotide sequence of the primer so that the primer can bind thereto in the reverse transcription step described below.

[0050] The 5' and / or 3' adapters may also include index or barcode sequences for identifying unique RNA molecules in a sample.

[0051] The base length of the adapter used in this step is not particularly limited. The base lengths of the 5' adapter and the 3' adapter may be the same or different. The base length of the adapter may be, for example, 10 to 300 bases, 20 to 200 bases, or 30 to 150 bases. Preferably, it may be 40 to 100 bases, 50 to 80 bases, or 60 to 70 bases.

[0052] This step yields adaptor-linked RNA, in which an adaptor is linked to the 5' and / or 3' end of the RNA. The adaptor ligation adds binding sequences for the primers used in the reverse transcription and amplification steps described below, enabling efficient reverse transcription and nucleic acid amplification.

[0053] (Reverse transcription step) The "reverse transcription step" is a step in which the adapter-linked RNA obtained in the adapter ligation step is reverse transcribed to obtain a reverse transcription product. The reverse transcription step is a step in which the adapter-linked RNA obtained in the adapter ligation step is used as a template to generate a reverse transcription product (cDNA) using a primer and an enzyme such as reverse transcription polymerase.

[0054] The primer used in this step can be a primer having a sequence complementary to at least a portion of the adaptor-linked RNA, preferably to at least a portion of the 3' adaptor linked to the 3' end of the RNA in the adaptor-linking step.

[0055] The reverse transcription method used in this step can be any method known in the art, for example, the reverse transcription method described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press.

[0056] (Amplification step) The "amplification step" is a step in which the reverse transcription product obtained in the reverse transcription step is amplified by a nucleic acid amplification method to obtain an amplified product.

[0057] As used herein, the term "nucleic acid amplification method" refers to a method of amplifying nucleic acids with a nucleic acid polymerase using primers. Examples include PCR, NASBA (Nucleic Acid Sequence-Based Amplification), ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids) (isothermal gene amplification), and LAMP (Loop-Mediated Isothermal Amplification) (registered trademark). The nucleic acid amplification method used in the present invention is not limited, but is preferably PCR, which typically uses a thermostable DNA polymerase.

[0058] The primer used in this step can have a sequence complementary to at least a portion of the reverse transcription product, preferably to at least a portion of the primer used in the reverse transcription step, so that the reverse transcription product (cDNA) obtained in the reverse transcription step can be amplified.

[0059] The primers may contain different index or barcode sequences for each sample, enabling low-cost, high-throughput multiplex analysis in which libraries of nucleic acid molecules from different samples are pooled and sequenced simultaneously by next-generation sequencing.

[0060] Nucleic acid amplification methods are well known in the art, and can be performed by referring to the conditions described in various protocols. Examples of protocol collections include the aforementioned Green, MR and Sambrook, J, (2012), Domingues L. (2017) PCR: Methods and Protocols, Methods in Molecular Biology, Humana Press, and Park DJ, (2010) PCR Protocols, Methods in Molecular Biology, Third Edition, Humana Press. Nucleic acid amplification kits are also commercially available from life science manufacturers, and these can also be used. In such cases, the conditions for nucleic acid amplification can be determined by following the attached instructions or the protocols recommended by each manufacturer.

[0061] (Acquisition process) The "obtaining step" refers to the step of obtaining a target length from the amplification products of various lengths amplified in the amplification step. Base length range This is a step of obtaining a nucleic acid molecule. Here, the "length of the target range" refers to the region of the amplification product excluding the adapter portion, m The length is determined so as to include both nucleic acid molecules derived from iRNA and nucleic acid molecules derived from miRNA precursors, and the specific length is 15 to 33 bases. In the present invention, unless otherwise specified, when the base length of a nucleic acid molecule is stated, it refers to the base length excluding the adapter portion.

[0062] In this process, Base length rangeThe phrase "obtaining a nucleic acid molecule" means that when obtaining nucleic acid molecules (nucleotide fragments) having a predetermined length from the amplification products, the nucleic acid molecules are obtained from the amplification products so that the desired length range of 15 to 33 bases is always included in the obtained range. Therefore, the base length range of the nucleic acid molecules obtained in this step is not limited as long as it is 15 bases or less, and is not limited as long as it is 33 bases or more. Examples of the base length range of the nucleic acid molecules to be obtained include 15 to 33 bases, 15 to 34 bases, 15 to 35 bases, 15 to 36 bases, 15 to 37 bases, 15 to 38 bases, 15 to 39 bases, 15 to 40 bases, 15 to 45 bases, 15 to 46 bases, 15 to 50 bases, 15 to 55 bases, 15 to 60 bases, 15 to 65 bases, 15 to 70 bases, 15 to 75 bases, and 15 to 80 bases. base length, 15-80 bases, 15-85 bases, 15-90 bases, 15-95 bases, 15-100 bases, 15-110 bases, 15-120 bases, 15-130 bases, 15-140 bases, 15-150 bases, 15-160 bases, 15-170 bases, 15-180 bases, 15-190 bases, 15-200 bases, 15-300 bases, or 15-1000 base length; 14-33 base length, 14-34 base length, 14-35 base length, 14-36 base length, 14-37 base length, 14-38 base length, 14-39 base length, 14-40 base length, 14-45 base length, 14-46 base length, 14-50 base length, 14-55 base length, 14-60 base length, 14-65 base length, 14-70 base length, 14-75 base length, 14-80 base length , 14-85 bases long, 14-90 bases long, 14-95 bases long, 14-100 bases long, 14-110 bases long, 14-120 bases long, 14-130 bases long, 14-140 bases long, 14-150 bases long, 14-160 bases long, 14-170 bases long, 14-180 bases long, 14-190 bases long, 14-200 bases long, 14-300 bases long, or 14-1000 bases long;13-33 base length, 13-34 base length, 13-35 base length, 13-36 base length, 13-37 base length, 13-38 base length, 13-39 base length, 13-40 base length, 13-45 base length, 13-46 base length, 13-50 base length, 13-55 base length, 13-60 base length, 13-65 base length, 13-70 base length, 13-75 base length, 13-80 base length, 13-85 base length, 13-90 base length, 13-95 base length, 13-100 base length, 13-110 base length, 13-120 base length, 13-130 base length, 13-140 base length, 13-150 Base length, 13-160 bases, 13-170 bases, 13-180 bases, 13-190 bases, 13-200 bases, 13-300 bases, or 13-1000 bases; 12-33 bases, 12-34 bases, 12-35 bases, 12-36 bases, 12-37 bases, 12-38 bases, 12-39 bases, 12-40 bases, 12-45 bases, 12-46 bases, 12-50 bases, 12-55 bases, 12-60 bases, 12-65 bases, 12-70 bases, 12-75 bases, 12-80 bases, 12-8 5 bases, 12-90 bases, 12-95 bases, 12-100 bases, 12-110 bases, 12-120 bases, 12-130 bases, 12-140 bases, 12-150 bases, 12-160 bases, 12-170 bases, 12-180 bases, 12-190 bases, 12-200 bases, 12-300 bases, or 12-1000 bases; 10-33 bases, 10-34 bases, 10-35 bases, 10-36 bases, 10-37 bases, 10-38 bases, 10-39 bases, 10-40 bases, 10-45 bases Base length, 10-46 bases, 10-50 bases, 10-55 bases, 10-60 bases, 10-65 bases, 10-70 bases, 10-75 bases, 10-80 bases, 10-85 bases, 10-90 bases, 10-95 bases, 10-100 bases, 10-110 bases, 10-120 bases, 10-130 bases, 10-140 bases, 10-150 bases, 10-160 bases, 10-170 bases, 10-180 bases, 10-190 bases, 10-200 bases, 10-300 bases, or 10-1000 bases;5-33 base length, 5-34 base length, 5-35 base length, 5-36 base length, 5-37 base length, 5-38 base length, 5-39 base length, 5-40 base length, 5-45 base length, 5-46 base length, 5-50 base length, 5-55 base length, 5-60 base length, 5-65 base length, 5-70 base length, 5-75 base length, 5-80 base length, 5-85 base length , 5 to 90 bases long, 5 to 95 bases long, 5 to 100 bases long, 5 to 110 bases long, 5 to 120 bases long, 5 to 130 bases long, 5 to 140 bases long, 5 to 150 bases long, 5 to 160 bases long, 5 to 170 bases long, 5 to 180 bases long, 5 to 190 bases long, 5 to 200 bases long, 5 to 300 bases long, or 5 to 1000 bases long; or 1 to The length may be 33 bases, 1 to 34 bases, 1 to 35 bases, 1 to 36 bases, 1 to 37 bases, 1 to 38 bases, 1 to 39 bases, 1 to 40 bases, 1 to 45 bases, 1 to 46 bases, 1 to 50 bases, 1 to 55 bases, 1 to 60 bases, 1 to 65 bases, 1 to 70 bases, 1 to 75 bases, 1 to 80 bases, 1 to 85 bases, 1 to 90 bases, 1 to 95 bases, 1 to 100 bases, 1 to 110 bases, 1 to 120 bases, 1 to 130 bases, 1 to 140 bases, 1 to 150 bases, 1 to 160 bases, 1 to 170 bases, 1 to 180 bases, 1 to 190 bases, 1 to 200 bases, 1 to 300 bases, or 1 to 1000 bases. ;

[0063] In one embodiment, the length of the nucleic acid molecule obtained in the obtaining step, excluding the adaptor portion, may be 100 bases or less. For example, the length, excluding the adaptor portion, may be 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, or 40 bases or less.

[0064] The range of the base length L of the nucleic acid molecule obtained in this step excluding the adaptor portion is determined by the following inequality, where Lmin represents the lower limit of the base length excluding the adaptor portion, and Lmax represents the upper limit of the base length excluding the adaptor portion: Lmin≦L≦Lmax (1) (wherein Lmin is 15 or less, and Lmax is 33 or more) In the above inequality (1), Lmin may be 15 or less, for example, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and / or Lmax may be 33 or more, for example, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, or 1000.

[0065] In one embodiment, the Lmax may be equal to or less than 100. For example, Lmax may be equal to or less than 90, 80, 70, 60, 50, or 40.

[0066] The range of nucleic acid molecules obtained in this step is exemplified in Figure 1. In Figure 1, some of the range of nucleic acid molecules obtained in the obtaining step is illustrated by the double-headed arrow on the right side of the figure. The nucleic acid molecules obtained include those with a base length of 15 to 33 bases, excluding the adaptor portion.

[0067] (Sequencing step) The "nucleotide sequence determination step" is a step of determining the nucleotide sequence of the nucleic acid molecule obtained in the obtaining step.

[0068] The nucleic acid molecules obtained after the obtaining step include a length in the desired range. Base length range This is a nucleic acid molecule. In this step, the base sequence of this nucleic acid molecule is determined.

[0069] Nucleic acid molecules to be subjected to base sequencing can be purified as necessary. Various nucleic acid purification methods known in the art can be used to purify nucleic acid molecules. Examples include separation and extraction methods using gel electrophoresis, adsorption purification methods using silica matrices or silica membranes, and column purification methods. Commercially available nucleic acid purification kits may be used to purify nucleic acids. In such cases, specific conditions for the nucleic acid purification method can be determined by following the attached kit or the protocol recommended by each manufacturer.

[0070] The base sequence of the amplification product may be determined by a base sequence determination method known in the art. Examples of base sequence determination methods include the Sanger method (dideoxy method) and next-generation sequencing. Next-generation sequencing may be a second-generation sequencing method or a third-generation sequencing method. Examples of second-generation sequencing methods include pyrosequencing, synthetic sequencing, ligation sequencing, and ion semiconductor sequencing. Examples of third-generation sequencing methods include sequencing methods using single-molecule sequencers or nanopore sequencers. These methods are known in the art, and various protocol collections and methods disclosed on the websites of various companies can be used as references.

[0071] In determining the base sequences in this step, it is preferable to use a next-generation sequencing method that can simultaneously determine the base sequences of multiple nucleic acid molecules.

[0072] (Measurement process) The "measurement step" is a step of measuring the number of reads of the target miRNA, its precursor, and / or its fragment. When the base sequence is determined by next-generation sequencing, this step can be included as a selection step.

[0073] When sequencing is performed using next-generation sequencing, the reads obtained in the sequencing process can be annotated and assigned to each miRNA gene, and can be classified as mature miRNAs, miRNA precursors, or fragments thereof. For annotation, for example, a miRNA sequence database (miRBase) can be used.

[0074] When comparing the number of reads of the target miRNA, its precursor, and / or its fragment obtained in this step among multiple samples, it is preferable to normalize them. The normalization coefficient used for normalization is not limited as long as it can be considered constant among the multiple samples being compared. For example, the number of reads of miRNAs that can be considered to have no expression fluctuations can be used as the normalization coefficient for each sample.

[0075] The target miRNA, its precursor, and / or its fragment for which the number of reads is measured in this step is not limited to a specific type. In one embodiment, the target miRNA may be one or more selected from the group consisting of hsa-miR-16-5p, hsa-miR-483-3p, hsa-miR-4492, hsa-miR-3648-1, and hsa-miR-663a.

[0076] 1-4.Effects In the detection method of this embodiment, in the obtaining step, a target length range corresponding to 15 to 33 bases is obtained. Base length range By obtaining nucleic acid molecules, it is possible to obtain both nucleic acid molecules derived from miRNAs and nucleic acid molecules derived from miRNA precursors, which allows for highly accurate detection of miRNAs and miRNA precursors even in samples containing very small amounts of miRNA.

[0077] Furthermore, in the detection method of this embodiment, by obtaining nucleic acid molecules that contain not only mature miRNAs but also miRNA precursors, it is possible to detect miRNAs and their precursors that can serve as biomarkers even from samples containing very small amounts of miRNA, making it possible to detect differences between samples even in samples containing very small amounts of miRNA.

[0078] 2. Method for detecting target miRNA and its precursor (without reverse transcription step) 2-1. Overview The first aspect of the present invention is a method for detecting a target miRNA and its precursor. This detection method includes, as essential steps, an RNA extraction step, an acquisition step, and a base sequencing step, and can detect a target miRNA and its precursor with high accuracy from samples containing very small amounts of miRNA, such as serum. This detection method differs from the detection method of the first aspect in that it does not include a reverse transcription step and instead subjects the RNA to base sequencing.

[0079] 2-2. Method The detection method of this embodiment includes an RNA extraction step, an acquisition step, and a base sequencing step as essential steps, and includes a measurement step as a selection step.

[0080] (RNA extraction process) The "RNA extraction step" refers to a step of extracting RNA from a sample. This step is similar to the description of the RNA extraction step of the first embodiment.

[0081] (Acquisition process) The "obtaining step" refers to the step of obtaining a fragment of RNA having a length in the desired range from the RNA extracted in the RNA extraction step. Base length range This is a process for obtaining nucleic acid molecules. Here, the "length within the desired range" is determined so as to fall within the range in which both miRNA and its precursor are obtained, and specifically, is a length of 15 to 33 bases.

[0082] In this process, Base length range "Obtaining nucleic acid molecules" means obtaining nucleic acid molecules so that the obtained range includes nucleic acid molecules with a length of 15 to 33 bases. Therefore, the range of base lengths of the nucleic acid molecules obtained in this step is not limited, with a lower limit of 15 bases or less and an upper limit of 33 bases or more.

[0083] Examples of the range of base lengths of the nucleic acid molecules to be obtained are the same as the range exemplified as the "length excluding the adaptor portion" in the first embodiment, and therefore will not be described here.

[0084] In one embodiment, the length of the nucleic acid molecule obtained in the obtaining step may be 100 bases or less.

[0085] (Sequencing step) The "nucleotide sequence determination step" is a step of determining the nucleotide sequence of the nucleic acid molecule obtained in the obtaining step. Since the nucleic acid molecule obtained in the obtaining step is RNA, the nucleotide sequence of the RNA is determined in this step.

[0086] In one embodiment, the base sequence of an RNA molecule is determined using a direct RNA sequencing method. Examples of direct RNA sequencing methods include nanopore sequencing using MinION, PromethION, GridION, Flongle, SmidgION, etc., provided by Oxford Nanopore Technology. These methods are well known in the art, and various protocol collections and methods disclosed on the websites of each company can be used as references.

[0087] RNA to be subjected to base sequencing may be purified as necessary. RNA purification can be performed using various nucleic acid purification methods known in the art. Examples include separation and extraction methods using gel electrophoresis, adsorption purification methods using silica matrices or silica membranes, and column purification methods. Commercially available nucleic acid purification kits may be used to purify nucleic acids. In such cases, specific conditions for nucleic acid purification can be determined by following the attached kit or the protocol recommended by each manufacturer.

[0088] (Measurement process) The "measuring step" is a step of measuring the number of reads of the target miRNA, its precursor, and / or its fragment.

[0089] The target miRNA, its precursor, and / or its fragment for which the number of reads is measured in this step is not limited to a specific type. In one embodiment, the target miRNA may be one or more selected from the group consisting of hsa-miR-16-5p, hsa-miR-483-3p, hsa-miR-4492, hsa-miR-3648-1, and hsa-miR-663a.

[0090] In this step, normalization can be performed in the same manner as in the first embodiment.

[0091] 2-3.Effects According to the detection method of this embodiment, the sequence of the RNA molecule itself can be read without reverse transcription, and therefore the number of reads of the target miRNA, its precursor, and / or its fragment can be measured without the bias of reverse transcription or PCR.

[0092] 3. Methods for assisting in disease diagnosis (when reverse transcription step is included) Overview The third aspect of the present invention is a method for assisting in the diagnosis of disease. The method of this aspect includes, as essential steps, an RNA extraction step, an adapter ligation step, a reverse transcription step, an amplification step, an acquisition step, a base sequencing step, and a determination step. This method can detect target miRNAs and their precursors with high accuracy from samples containing very small amounts of miRNA, such as serum, and determine whether a subject has a disease. The method of this aspect differs from the method of the fourth aspect in that it includes a reverse transcription step and subjects the nucleic acid after the reverse transcription step to base sequencing.

[0093] 3-2.Definition of Terms As used herein, the term "disease" is not limited to, but includes, for example, cancer, hypertension, diabetes, heart disease, cerebrovascular disease, neuropsychiatric disorders, immune / allergic diseases, infectious diseases, etc. In this embodiment, the disease is preferably cancer.

[0094] As used herein, the type of "cancer" is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, glioma, pediatric tumors such as neuroblastoma, leukemia, and lymphoma. The cancer is preferably pancreatic cancer or breast cancer.

[0095] In this embodiment, the "control" refers to a healthy individual, i.e., an individual not suffering from a specific disease, preferably an individual not suffering from any disease. The control may be one individual or multiple individuals.

[0096] 3-3. Method The method of this embodiment includes, as essential steps, an RNA extraction step, an adaptor ligation step, a reverse transcription step, an amplification step, an acquisition step, a base sequencing step, a measurement step, and an evaluation step.

[0097] The RNA extraction step, adapter ligation step, reverse transcription step, amplification step, acquisition step, measurement step, and base sequencing step in the method of this embodiment are the same as those in the first embodiment, except that samples derived from a subject and a control are used. Therefore, the determination step will be described below.

[0098] (Judgment process) The determination step is a step of comparing the number of reads of the subject and the control obtained in the measurement step, and determining that the subject is suffering from a disease if the number of reads of the subject is different from the number of reads of the control.

[0099] In one embodiment, the determination step compares the number of reads obtained in the measurement step between the subject and the control, and if the number of reads in the subject is greater than the number of reads in the control, it is determined that the subject is suffering from a disease.

[0100] In another embodiment, the determination step compares the number of reads obtained in the measurement step between the subject and the control, and if the number of reads in the subject is smaller than the number of reads in the control, the subject is determined to be suffering from a disease.

[0101] In this step, the "number of reads from a subject" refers to the number of reads from the target miRNA, its precursor, and / or its fragment obtained from the subject. Preferably, the number of reads from a subject is the number of reads from the target miRNA and its precursor obtained from the subject, or the number of reads from the target miRNA, its precursor, and its fragment obtained from the subject. The same applies to the "number of reads from a control."

[0102] In this embodiment, the method for comparing the read numbers of a subject and a control is not limited. For example, a cutoff value for the read number of a subject is determined based on the read number of the control, and the comparison is made based on the cutoff value. That is, a predetermined value is determined as the cutoff value, and if the measured value is equal to or greater than (or equal to or less than) the read number of the subject, it can be determined that the read number of the subject is greater than (or smaller than) the read number of the control.

[0103] The cutoff value refers to a boundary value for comparing the number of reads in a subject and a control. The cutoff value can usually be calculated based on the disease incidence rate and the sensitivity and specificity calculated from a receiver operating characteristic curve (ROC curve). There are no particular limitations on the method for setting the cutoff value.

[0104] For example, the number of reads in healthy individuals not affected by the disease, or the average number of reads in a group of healthy individuals, can be used as a cutoff value, and when the number of reads in a subject is higher (or lower) than the cutoff value, it can be determined that the number of reads in the subject is greater (or smaller) than the number of reads in the control.

[0105] Alternatively, the number of reads in a subject can be determined to be greater than the number of reads in a control when the number of reads in the subject is higher than a cutoff value set at 1.5 times or more, 2.0 times or more, 3.0 times or more, 4 times or more, 5 times or more, or 6 times or more the number of reads in healthy subjects not affected by the disease or the average number of reads in a group of healthy subjects. Alternatively, the number of reads in a subject can be determined to be smaller than the number of reads in a control when the number of reads in the subject is lower than a cutoff value set at 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less the number of reads in healthy subjects not affected by the disease or the average number of reads in a group of healthy subjects.

[0106] Alternatively, the measured values ​​obtained from the control group can be classified by percentile, and the percentile value used for the classification can be used as the cutoff value. For example, the 95th percentile of the number of reads obtained from the control can be used as the cutoff value, and if the number of reads of the subject is equal to or greater than the 95th percentile, it can be determined that the number of reads of the subject is greater than the number of reads of the control. Alternatively, the 5th percentile of the number of reads obtained from the control can be used as the cutoff value, and if the number of reads of the subject is equal to or less than the 5th percentile, it can be determined that the number of reads of the subject is smaller than the number of reads of the control.

[0107] The lead number of the control healthy subject is different from the lead number of the subject, and does not necessarily need to be measured each time. For example, if the amount of sample used for measurement, the measurement method, and the measurement conditions are kept constant, the lead number of the control healthy subject measured previously can be reused.

[0108] Effects In this embodiment, by obtaining nucleic acid molecules that contain not only mature miRNAs but also miRNA precursors, it is possible to detect miRNAs and their precursors that can serve as biomarkers even from samples containing very small amounts of miRNA, and by detecting differences between samples, it is possible to assist in the differentiation of disease.

[0109] 4. Methods for assisting in disease diagnosis (without reverse transcription step) Overview The fourth aspect of the present invention is a method for assisting in the diagnosis of disease. The method of this aspect includes, as essential steps, an RNA extraction step, an acquisition step, a base sequencing step, and a determination step. It can detect target miRNAs and their precursors with high accuracy from samples containing very small amounts of miRNA, such as serum, and determine whether a subject has a disease. The method of this aspect differs from the method of the third aspect in that it does not include a reverse transcription step and instead subjects RNA to base sequencing.

[0110] 4-2. Method The method of this embodiment includes, as essential steps, an RNA extraction step, an acquisition step, a base sequencing step, a measurement step, and an evaluation step.

[0111] The RNA extraction step, acquisition step, measurement step, and base sequencing step in the method of this embodiment are the same as those in the second embodiment, except that samples derived from a subject and a control are used. The determination step in the method of this embodiment is the same as that in the third embodiment.

[0112] Effects According to the method of this embodiment, the sequence of the RNA molecule itself is read without reverse transcription, so the number of reads of the target miRNA, its precursor, and / or its fragment can be measured without the bias of reverse transcription or PCR, thereby assisting in the diagnosis of disease. [Example]

[0113] The present invention will be specifically described below with reference to examples, but these examples are merely illustrative and the present invention is not limited to the scope described in the examples.

[0114] Example 1: Detection of miRNAs and their precursors in the serum of breast cancer patients (the purpose) We will detect miRNAs and their precursors in serum collected from breast cancer patients and healthy individuals using next-generation sequencing. Furthermore, we will identify miRNA genes that can distinguish between cancer and healthy samples based on the difference in the amount of miRNAs and their precursors detected in serum.

[0115] (Methods and Results) (1) Preparation of sequencing library Serum samples were collected from five breast cancer patients and five healthy subjects.

[0116] Total RNA was extracted from 300 μL of serum using 3D-Gene RNA extraction reagent from liquid sample.

[0117] Next, a sequencing library was prepared from the total RNA using the TruSeq small RNA library prep kit (Illumina, Inc.). After ligating 5' and 3' adapters to both ends of the RNA molecules, single-stranded cDNA was synthesized by reverse transcription using an RT primer that recognized the 3' adapter sequence. PCR amplification was performed using the resulting single-stranded cDNA as a template using a PCR primer pair with index sequences (total length of the primer pair was 130 bases, each primer contained a 55-base Read1 sequence and a 63-base Read2 sequence).

[0118] The PCR products were electrophoresed using BluePippin, and after ethidium bromide staining, the gel was excised from the range of approximately 145 to 430 bases (corresponding to the range of 22 to 300 bases excluding the adapter sequence). DNA was extracted from the excised gel and used as a sequencing library.

[0119] (2) Expression analysis using next-generation sequencing Using the next-generation sequencer "Illumina Hiseq" (Illumina Inc.), base sequence data was obtained by reading 100 bases using the paired-end method. The obtained base sequence data was read-trimmed and reads of identical sequences were counted. In addition, the data was mapped to the human genome sequence and annotated based on the miRNA sequence database (miRBase). The read sequences assigned to each miRNA were counted, and the expression levels of mature miRNAs and miRNA precursors in each sample were quantified.

[0120] Next, the quantified known miRNAs were normalized using the RPM normalization method and the iterative differentially expressed genes or transcripts elimination strategy (iDEGES). Using the statistical analysis software R, edgeR (with replicates) or DESeq (without replicates) was used to detect DEGs. A normalization coefficient was determined three times using only the expression information of genes determined to be non-DEGs. Genes with significantly different expression levels between different groups were identified using the read counts normalized by this normalization coefficient. For analysis, we used the statistical analysis software R 3.6.2 (R Development Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, URL http: / / www.R-project.org / ) and the differentially expressed gene detection package "TCC" provided by R.

[0121] (3) Identification of discriminant genes Using the normalized read counts for each miRNA obtained in (2), (a) the expression level of mature miRNA, (b) the expression level of precursor miRNA, and (c) the sum of the expression levels of mature miRNA and precursor miRNA were calculated for each of the cancer and healthy specimens. For each of (a) to (c), a two-tailed t-test assuming equal variance was used to calculate p-values ​​between the cancer and healthy specimens. Three miRNA genes, hsa-miR-16-5p (SEQ ID NO: 1), hsa-miR-483-3p (SEQ ID NO: 2), and hsa-miR-4492 (SEQ ID NO: 3), were identified as discriminant genes with p-values ​​of less than 0.05 and statistical significance.

[0122] When either (a) the expression level of the mature miRNA or (b) the expression level of the precursor miRNA was used alone, hsa-miR-16-5p was unable to distinguish between cancer and healthy samples (Figure 2). On the other hand, when (c) the combined expression level of the mature miRNA and the precursor miRNA was used, it was possible to distinguish between cancer and healthy samples (Figure 2).

[0123] The base sequences of the mature hsa-miR-16-5p and its precursor, as well as examples of the base sequences detected in this example, are shown in Table 1 below.

[0124] [Table 1]

[0125] The base sequences of the mature hsa-miR-483-3p and its precursor, as well as examples of the base sequences detected in this example, are shown in Table 2 below.

[0126] [Table 2]

[0127] The base sequences of the mature hsa-miR-4492 and its precursor, as well as examples of the base sequences detected in this example, are shown in Table 3 below.

[0128] [Table 3]

[0129] Example 2: Detection of miRNA in serum of pancreatic cancer patients (the purpose) We will detect miRNAs and their precursors in serum collected from pancreatic cancer patients and healthy individuals using next-generation sequencing. Furthermore, we will identify miRNA genes that can distinguish between cancer and healthy individuals based on the difference in the amount of miRNAs and their precursors detected in serum.

[0130] (Methods and Results) Serum samples were collected from five pancreatic cancer patients and five healthy controls. Expression analysis of serum RNA was performed using next-generation sequencing, as in Example 1. Furthermore, as in Example 1, (a) the expression level of mature miRNA, (b) the expression level of precursor miRNA, and (c) the sum of the expression levels of mature miRNA and precursor miRNA were calculated for each of the cancer and healthy controls, and p-values ​​were calculated between the cancer and healthy controls. As a result, two miRNA genes, hsa-miR-3648-1 (SEQ ID NO: 4) and hsa-miR-663a (SEQ ID NO: 5), were identified as discriminant genes with p-values ​​of less than 0.05 and statistical significance.

[0131] When either (a) the expression level of mature miRNA or (b) the expression level of precursor miRNA was used alone, hsa-miR-3648-1 and hsa-miR-663a were unable to distinguish between cancer and healthy samples. On the other hand, when (c) the combined expression level of mature miRNA and precursor miRNA was used, cancer and healthy samples could be distinguished.

[0132] These results demonstrate that when targeting samples containing very small amounts of miRNA, such as serum, differences in expression levels between specimens can be detected by preparing nucleic acid molecules that contain both mature and precursor miRNAs. Furthermore, it was demonstrated that disease can be identified by using the combined expression levels of mature and precursor miRNAs.

[0133] The base sequences of the mature form of hsa-miR-3648-1 and its precursor, as well as examples of the base sequences detected in this example, are shown in Table 4 below.

[0134] [Table 4]

[0135] The base sequences of the mature hsa-miR-663a and its precursor, as well as examples of the base sequences detected in this example, are shown in Table 5 below.

[0136] [Table 5]

Claims

1. 1. A method for detecting a target miRNA and its precursor in a sample, comprising: an RNA extraction step of extracting RNA from the sample; an adapter ligation step of ligating an adapter to the 5' end and / or 3' end of the extracted RNA; a reverse transcription step of reverse transcribing the adapter-ligated RNA obtained in the adapter ligation step to obtain a reverse transcription product; an amplification step of amplifying the reverse transcription product by a nucleic acid amplification method to obtain an amplification product; An obtaining step of obtaining nucleic acid molecules having a base length range including a length range of interest from the amplification products; a base sequencing step of determining the base sequence of the nucleic acid molecule obtained in the obtaining step Including, The target length range is 15 to 33 bases long excluding the adaptor portion, The base length range of the nucleic acid molecule obtained in the obtaining step is such that the lower limit Lmin of the base length range excluding the adapter portion is 15 or less, and the upper limit Lmax of the base length range excluding the adapter portion is 100 or more. method.

2. The method described in claim 1, wherein the upper limit Lmax of the base length range is 200 or more.

3. The method according to claim 1 or 2, wherein the base sequence determination is performed using next-generation sequencing.

4. A measuring step for measuring the number of reads of the target miRNA, its precursor, and / or its fragment. The method of any one of claims 1 to 3, further comprising:

5. The method according to any one of claims 1 to 4, wherein the sample is a body fluid.

6. A method for assisting in the diagnosis of a disease, comprising: an RNA extraction step for extracting RNA from body fluids derived from the subject and the control; an adapter ligation step of ligating an adapter to the 5' end and / or 3' end of the extracted RNA; a reverse transcription step of reverse transcribing the adapter-ligated RNA obtained in the adapter ligation step to obtain a reverse transcription product; an amplification step of amplifying the reverse transcription product by a nucleic acid amplification method to obtain an amplification product; an obtaining step of obtaining nucleic acid molecules having a base length range including a length range of interest from the amplification products; a base sequence determination step of determining the base sequence of the nucleic acid molecule obtained in the obtaining step by next-generation sequencing; Measuring the number of reads of the target miRNA, its precursor, and / or its fragment; and The number of reads in the subject and the control were compared, and the number of reads in the subject differed from the number of reads in the control. determining that the subject is suffering from a disease if The length of the target range is 15 to 33 bases excluding the adapter portion, The base length range of the nucleic acid molecule obtained in the obtaining step is such that the lower limit Lmin of the base length range excluding the adapter portion is 15 or less, and the upper limit Lmax of the base length range excluding the adapter portion is 100 or more. method.

7. The method of claim 6, wherein the control subject is a healthy subject.

8. 8. The method of claim 6 or 7, wherein the body fluid is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, interstitial fluid, and saliva.

9. The method according to any one of claims 6 to 8, wherein the disease is cancer.

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