The name of the invention: a method for evaluating the quality of body fluids derived from miRNA

Technical field

[0001]

　The present invention relates to a method for evaluating the quality of the miRNA from bodily fluid analyte.

Background technique

[0002]

　miRNA (micro-RNA) is, RNA (precursor) of the hairpin-like structure from genomic DNA coming transcribed as. This precursor, dsRNA cleavage enzyme (Drosha, Dicer) having a specific enzyme RNase III cleavage activity after being cut by, changes to the form of double-stranded, then a single strand. Then, incorporated into a protein complex antisense strand of one is referred to as a RISC, are thought to be involved in mRNA translation inhibition. Thus, miRNA after transfer, the embodiment because different at each step, usually, if the detection target an miRNA hairpin structure, a double-stranded structure, various forms such as single chain structure it is necessary to take into account. miRNA comprises 15 to 25 bases of RNA, and its presence is confirmed in a variety of organisms.

[0003]

　Recently, miRNA not only the cell, serum is the sample containing no cells, plasma, urine, spinal fluid or the like body fluid and there are many, the expression level of various diseases, including cancer It could become a biomarker has been suggested. miRNA is 2016 February, there are more than 2500 species in humans, when using a measurement system such as a high sensitivity DNA microarrays to detect simultaneously the 1000 or expression of miRNA exceeding of which in serum and plasma It is possible. Thus, serum and plasma using a DNA microarray method, urine, biomarker discovery study of body fluid of the spinal fluid and the like are carried out, expand into bio-marker test that can discover the disease at an early stage is expected .

[0004]

　Meanwhile, RNA is heat or enzymes, are easily decomposed substance by physical and chemical variety of factors, such as freeze-thaw, when performing gene expression analysis using a DNA microarray, RNA degradation amount of expression it is known that affect the measurement. As biomarkers of the disease, in a test that measures the expression level of miRNA contained in body fluids, the results in testing and diagnosis based on the measurement of the expression amount with uncertainty, missed opportunities for appropriate treatment or, it will be or strong wrong unnecessary economic to the patient in applying the medical, physical fitness burden. Therefore, in order to accurately measure the expression level, it is very important that miRNA to test target is used to inspect the specimen is not decomposed.

[0005]

　Conventionally, as a method of measuring the resolution of RNA, generally it has electrophoresis is used, for example, can be determined from the concentration ratio of 28S ribosomal RNA from the band and 18S ribosomal RNA from the band (28S / 18S) . Further, that as another approach, the difference in length of the RNA segments Patent Document 1, a method for quantitatively evaluating the degree of degradation RNA have been proposed, where the segment length is shortened when the nucleotide is decomposed, We are using the features of the long-chain RNA.

[0006]

　However, when measuring the expression level of miRNA is, for utilizing the short chain fractions RNA many, in this case does not contain long chain RNA, conventional method as described above, measuring the degree of degradation RNA It is an effective approach to not become. From the correlation coefficients of all genes in the gene expression analysis results, can also be measured resolution of RNA using the data of all genes is required, it takes time and effort. Therefore, paying attention to the decomposition fragments from long chain RNA, as an index of degradation fragments mixed in short chain fraction, a method of evaluating a degree of degradation miRNA in short chain fraction has been developed (Patent Document 2) .

CITATION

Patent Document

[0007]

Patent Document 1: JP-T 2015-519045 Patent Publication
Patent Document 2: JP 2008-35779 JP

Summary of the Invention

Problems that the Invention is to Solve

[0008]

　As described above, in order to accurately measure the expression amount of RNA that target, it is important to evaluate the quality by measuring the decomposition degree of RNA in the sample. However, the conventional method described above is where a method using ribosomal RNA or long RNA, ribosomal RNA or long RNA is an RNA present in the nuclear and cytoplasmic, such as serum, plasma, urine, almost non-existent in a body fluid sample of spinal fluid and the like. Therefore, this by the conventional methods, it was not possible to evaluate the quality degradation of the miRNA contained in the body fluid sample to accurately measure.

[0009]

　An object of the present invention, as a sample, in the case of using a body fluid sample unsuitable for conventional methods, is to find a method to evaluate the quality by measuring the decomposition degree of miRNA contained in the body fluid sample.

Means for Solving the Problems

[0010]

　To solve the above problems, the present inventors have, miRNA amount present depending on the decomposition of the nucleic acid sample contained in a body fluid sample is varied (hereinafter, referred to as "reference miRNA".) Measuring the abundance relative to the doing, it found that it is possible to evaluate the quality of the miRNA that target, thereby completing the present invention. That is, the present invention is to at least one reference miRNA of miRNA shown in SEQ ID NO: 1-12, standard bodily fluid analyte is a state in which the abundance of the reference miRNA in the body fluid sample, degradation of the nucleic acid sample is not in progress by comparing the abundance of miRNA in a method of assessing the quality of the miRNA, including the following aspects.

[0011]

(1) A method for evaluating the quality of the miRNA from bodily fluid analyte,
　prepared from the body fluid sample and standard bodily fluid analyte, by using the RNA samples containing miRNA, consisting of the nucleotide sequence represented by SEQ ID NO: 1-12 of one or more reference miRNA is selected from the miRNA, to measure the abundance in body fluid sample and standard bodily fluid analyte respectively, the measuring step;
　abundance of one or more criteria miRNA in the body fluid analyte measurement value or the representative value and compared to the abundance measurements or representative value of 1 or more criteria miRNA in standard bodily fluid analyte, abundance measurements or representative of one or more criteria miRNA between bodily fluid analyte and standard bodily fluid analyte obtaining a difference or ratio values, comparing step; and
　obtained in the comparing step, one or abundance measurements of a plurality of reference miRNA or based on the difference or ratio of the representative value, the quality of the miRNA from bodily fluid analyte determining the quality, determining step
including, Serial method.
(2) the step of comparing the difference or ratio of the abundance measurements of first reference miRNA, difference or ratio of the abundance measurements of each of the plurality of reference miRNA, or representative values of abundance measurements of a plurality of reference miRNA a step of obtaining a difference or ratio, (1) the method according.
(3) the determination step comprises comparing the difference or ratio of the abundance measurements or the representative value of the one or more reference miRNA, with a predetermined threshold value as a reference, (1) or (2 ) the method described.
(4) the step of comparing, determining a difference by subtracting the abundance measurements or the representative value in the standard bodily fluid analyte from abundance measurements or the representative value in the body fluid sample, or the presence in a body fluid sample the amount measured value or the representative value is divided by abundance measurements or the representative value in the standard bodily fluid analyte comprises determining the ratio, the method according to any one of (1) to (3).
(5) the determination step comprises comparing the difference or ratio of the abundance measurements or the representative value of the one or more reference miRNA, with a predetermined threshold value as a reference, the difference or ratio is the It determines that good quality miRNA derived bodily fluid analyte if it exceeds the threshold value, (4) the method according.
(6) the representative value of the abundance measurements of a plurality of reference miRNA bodily fluid analyte and during normal body fluid specimen, respectively, an average value or median abundance measurements of a plurality of reference miRNA, (1) ~ ( the method according to any one of 5).
(7) the measuring step includes correcting the abundance measurements of one or more criteria miRNA in a body fluid sample and standard bodily fluid analyte, subsequent steps are performed using the corrected measurements, (1) the method according to any one of - (6).
(8) the measuring step, a probe for capturing an immobilized the one or more reference miRNA on a support, and a nucleic acid sample extracted from a bodily fluid sample and standard bodily fluid analyte labeled with a labeling substance by performing hybridization in contact respectively comprises measuring the abundance of the one or more criteria miRNA bodily fluid analyte and during normal bodily fluid analyte, according to any one of (1) to (7) the method of.
(9) the measuring step, at the same time as the measurement of the abundance of the one or more reference miRNA in the body fluid sample comprises measuring the abundance of a target miRNA in the body fluid sample, (1) - (8 the method according to any one of).
(10) said measuring step comprises correcting the abundance measurements of the target miRNA in the body fluid sample, (9) The method according.
(11) said measuring step has been immobilized on a support, it is extracted and probe for capturing the probe and the one or more reference miRNA for capturing a target miRNA, from bodily fluid analyte labeled with a labeling substance by contacting the nucleic acid sample that has been performing hybridization comprises measuring in a body fluid sample target miRNA and the one or more abundance of reference miRNA, respectively, (9) or (10) described Method.
(12) the body fluid sample is blood, serum or plasma, (1) The method according to any one of - (11).
(13) In order to evaluate the quality of the miRNA from bodily fluid analyte, one or more computers,
　were prepared from the body fluid sample and standard bodily fluid analyte were measured respectively using the RNA samples containing miRNA, SEQ ID NO: 1 acquires abundance measurements in body fluid sample and standard bodily fluid analyte of one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by ~ 12, measured value acquisition step;
　in a body fluid sample of one or more the abundance measurements or the representative value of the reference miRNA, as compared to the abundance measurements or representative value of 1 or more criteria miRNA in standard bodily fluid analyte, one or more of between bodily fluid analyte and standard bodily fluid analyte and; of obtaining a difference or ratio of the abundance measurements or the representative value of the reference miRNA, comparing step
　difference or ratio of the obtained in the comparison step, one or more reference abundance measurements or the representative value of the miRNA on the basis of, from body fluids specimen determining the quality of the quality of the miRNA, determination step
program for executing.
(14) (13) was recorded the description of the program, a computer-readable recording medium.
(15) a probe for capturing one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1 to 12 comprising the immobilized support, miRNA quality evaluation chip.
(16) comprises a support probe is immobilized to capture one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by the probe and SEQ ID NO: 1-12 for capturing a target miRNA , chip for miRNA expression analysis.

Effect of the invention

[0012]

　The present invention, in the conventional method is difficult, it becomes possible quality evaluation of miRNA is contained in a body fluid sample. Further, according to the present invention, a body fluid specimen, for example, since miRNA to whether it has a quality suitable for gene expression analysis can be evaluated and conveniently a high precision using the expression amount of the biomarker in a body fluid sample the enables inspection of a more precise disorder as an indicator.

DESCRIPTION OF THE INVENTION

[0013]

　The present invention provides a method for evaluating the quality of the miRNA from bodily fluid analyte (degree of degradation), one or more miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1 to 12 as a reference miRNA the abundance of reference miRNA in the body fluid sample, the measuring step measures the abundance of reference miRNA in standard bodily fluid analyte; and measured or the representative value of the reference miRNA in body fluids samples obtained in the measuring step , measured values ​​or comparative obtaining a difference or ratio of the representative value of the reference miRNA contained in the standard bodily fluid analyte; based on the difference or ratio of the abundance measurements obtained in the comparison step, miRNA derived bodily fluid analyte determination step of determining quality (degree of degradation) of a method comprising the.

[0014]

　Abundance of reference miRNA in bodily fluid analyte can be examined by measuring the reference miRNA amount of RNA in the samples extracted from the bodily fluid sample. "The quality of miRNA contained in the body fluid specimen", the term "quality of miRNA in a body fluid specimen", "quality of miRNA from body fluids specimen", the term synonymous with the quality of the miRNA of RNA in the sample that was extracted from the body fluid specimen it is.

[0015]

　The method of the present invention, gene expression analysis, for example analysis and using array chip microarrays such as the polymerase chain reaction (PCR) method, in the analysis by sequencing, by evaluating the quality of miRNA contained in the body fluid sample in advance it can be used to determine the appropriateness of performing these analyzes. The gene expression analysis, for example, by labeling miRNA in the body fluid, the miRNA with a probe for capturing one or more target miRNA, a support and the probe is fixed for capturing a reference miRNA the expression level measuring, and primers for amplifying one or more target miRNA, perform amplification reaction using a primer for amplifying a reference miRNA, such as by measuring the expression level of a target miRNA It includes further analysis and testing of utilizing these results gene expression, for example, include carrying out the test to measure the clinical specimen in order to grasp the condition.

[0016]

　"MiRNA" is a type of non-coding RNA (ncRNA), which refers to a chain length of 15 to 25 bases about the short RNA produced in vivo, is believed to have the ability to modulate expression of mRNA. miRNA is, genomic DNA Caracalla hairpin-like structure RNA (precursor) come transcribed as. This precursor, dsRNA cleavage enzyme (Drosha, Dicer) having a specific enzyme RNase III cleavage activity after being cut by, changes to the form of double-stranded, then a single strand. Then, incorporated into a protein complex antisense strand of one is referred to as a RISC, are thought to be involved in mRNA translation inhibition. Thus, miRNA after transfer, since the embodiment differ in each stage, usually, when the miRNA target (detection target) is hairpin structure, a double-stranded structure, such as a single chain structure it is necessary to take into account the various forms. miRNA is its presence has been confirmed in a variety of organisms.

[0017]

　Bodily fluid analyte to which the present invention can be applied is a body fluid sample separated from a living body, such as blood, serum, plasma, urine, spinal fluid, saliva, swabs, there may be mentioned body fluids of various tissue fluid or the like, these the present invention is not limited to. Type of organism from which the body fluid sample is not particularly limited and various species are included, typically a mammal, especially a human.

[0018]

　Quality of miRNA in these body fluids is lowered, i.e. as a cause decompose miRNA, other such as temperature or heat, external forces such as vibration or ultrasound for body fluids, various direct including, such as electric field and magnetic field, indirect Although such physical force target is considered, but is not limited to these causes of degradation.

[0019]

　In the present invention, it is possible to extract the RNA from these specimens, measuring the expression level of miRNA using the RNA. RNA extraction, known methods (e.g., Favaloro et al method (Favaloro et al, Methods Enzymol 65:... 718 (1980)), etc.) and various commercially available kits for RNA extraction (for example, Qiagen Company of miRNeasy, Toray Industries, Inc. of "3D-Gene" RNA extraction reagent from liquid sample, etc.) can be applied.

[0020]

　In the present invention, abundance in a bodily fluid analyte of one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, and a base represented by SEQ ID NO: 1-12 to measure the abundance in the standard bodily fluid analyte of one or more reference miRNA is selected from the miRNA consisting of SEQ. Simultaneously with the measurement of the abundance of the reference miRNA in a body fluid sample, it may measure the abundance and correcting abundance of standard nucleic acid target miRNA to be described later.

[0021]

　Used as a reference miRNA in the present invention, miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12 is a miRNA that amount present depending on the degradation of the nucleic acid samples in a body fluid sample is selected as the miRNA to decrease. In general, when RNA is decomposed and partly for fragmenting the abundance of the gene RNA is reduced. In this case, the entire gene detected by gene expression analysis, correlation with RNA that are not degraded and degraded RNA decreases, for example, the correlation coefficient is 0.95 or less. Reference miRNA for use in the present invention is a miRNA that amount present in correlation with the degradation of such RNA varies (decreases). For example, the expression levels obtained by the correction processing shown later, the expression level ratio of the post-RNA degradation before and RNA degradation, preferably less than 0.8, more preferably miRNA such that 0.7 or less.

[0022]

　When using the serum (blood) as a body fluid sample, based miRNA for use in the present invention can amount present depending on the storage time at serum state is preferably selected to greater reduced miRNA. miRNA amount present depending on the storage time is reduced, for example, a serum sample was prepared after collection refrigerator (e.g., 4 ° C.) and stored at, after storage start 0 hours, 6 hours, 12 hours, 24 hours after further after every other day by measuring the abundance of miRNA after 7 days, to compare the extent of its reduction can be selected. If the period of storing the serum specimens in refrigerator up to more long-term, in accordance with the the period, for example by extending the storage time until after 2 weeks and 1 month, compared by measuring the abundance of miRNA good. Thus the abundance of miRNA obtained from different serum preserved time, by applying a statistical method, comparison between groups of gene expression analysis, the miRNA decrease in abundance is statistically significant over time it can be selected to. For example, generally based on such use is the t-test statistical analysis may be utilized. For example, package "SAM" Statistical Language "R" (Tusher VG et. Al., Proc Natl Acad Sic USA. 2001 98 (9) 5116-5121) can also be directly applied.

[0023]

　In the measurement process of the present invention, certain 12 types of the present inventors have selected miRNA, preferably 1 or more reference miRNA selected from 12 types of miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12 to measure the abundance of a body fluid sample and in the standard bodily fluid analyte. The standard bodily fluid analyte, a specimen degradation of the nucleic acid sample is not in progress, as compared to the bodily fluid analyte, a sample used as a reference for determining the acceptability of the quality of the miRNA contained in the body fluid sample. As the standard bodily fluid analyte, for example, those just after obtained or prepared bodily fluid analyte of interest, it can be degradation of the nucleic acid sample contained therein using the same specimen in a state where no progress. Or, if unable to obtain the target bodily fluid analyte the same sample just after preparation, it can also be used those prepared from another individual of the same species of fluid same species. Or it may be used to obtain the same species bodily fluid analyte which is commercially available as a standard. If the body fluid sample is serum (blood) it can be used specimens immediately after preparation of the serum specimen (specimen after storage time 0 hours elapsed) as the standard bodily fluid analyte. If serum specimens immediately after preparation is not available, may be used serum specimens immediately (after storage time 0 hours elapsed) prepared from another individual of the same species, may be used sera are commercially available .

[0024]

　Hereinafter, or reference miRNA, refers target miRNA to be described later, the probe for capturing the nucleic acid, such as correction standard nucleic acid are also collectively "capture probe" or simply "probe".

[0025]

　Determination of the presence of the miRNA, for instance, can be carried out by hybridization assays using array chip microarrays such as a probe immobilized on a support which specifically binds to a miRNA of interest. In the present invention, it is possible to capture one or more criteria miRNA is "reference miRNA capture probe" using an array chip containing immobilized support. Further, by using the array chip including a "target miRNA capture probe" support "for correction standard nucleic acid capture probe" is further immobilized to capture correction standard nucleic acid for capturing a target miRNA to be described later it may be.

[0026]

　The "capture probe" or "probe for capturing", directly or indirectly, and miRNA capturing object, preferably directly, and means a substance capable of selectively binding, as a typical example it can include nucleic acids, proteins, sugars and other antigenic compounds. In the present invention, it can be preferably used a nucleic acid probe. Nucleic acid, in addition to DNA and RNA, it is possible to use a nucleic acid derivative such as PNA (peptide nucleic acid), or LNA (Locked Nucleic Acid). Here, the derivative, in the case of nucleic acid, labeled derivatives due fluorophore, modified nucleotides (e.g., halogen, alkyl such as methyl, alkoxy such as methoxy, thio, nucleotides include groups such as carboxymethyl, and a base re configuration, the double bonds of the saturated, deamination, means a chemically modified derivatives such as derivatives containing nucleotides, etc.) that received such substitution of sulfur molecules oxygen molecules.

[0027]

　The chain length of the nucleic acid probe in order to ensure the stability and specificity of hybridization, it is preferably not less than the length of the miRNA to be detected. Usually, 17 if ~ 25 bases about the chain length, the probe can be sufficiently exhibited selective binding to miRNA of interest. Such chain length short oligo nucleic acid probes can be readily prepared by well known chemical synthesis method.

[0028]

　Nucleic acid probe, it is preferable that a completely complementary nucleotide sequence and miRNA of interest, even if there are differences in some, high homology to the extent capable of hybridizing with miRNA stringent conditions of the target base if sequences can be used as capture probes.

[0029]

　Hybridization time of stringency, temperature, salt concentration, chain length of the probe, is known to be a function of the concentration of the chaotropic agent in GC content and hybridization buffer nucleotide sequence of the probe. Stringent conditions, for example, Sambrook, J. et al (1998) Molecular Cloning:. (. 2nd ed) A Laboratory Manual, or the like can be used conditions described in Cold Spring Harbor Laboratory Press, New York . Stringent temperature conditions is about 30 ° C. or higher. Other conditions, hybridization time, detergents (e.g., SDS) concentration, and a presence or absence of carrier DNA and the like, by combining these conditions, it is possible to set various stringency. Those skilled in the art can appropriately determine the conditions for obtaining the function as capture probes were prepared for detection of the desired analyte RNA.

[0030]

　Sequence information of the miRNA, can be obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), such as a database or miRBase website (http://www.mirbase.org/) it can. Reference miRNA capture probe, the target miRNA capture probe and the correction standard nucleic acid capture probes can be designed based on sequence information available from those sites.

[0031]

　The number of miRNA capture probe that is immobilized on a support is not particularly limited. For example, the number of miRNA capture probe to cover all known miRNA that sequences have been identified using the one immobilized on a support, may measure the abundance of miRNA, for inspection purposes, etc. it may be used after immobilizing the desired number of miRNA capture probe on a support according.

[0032]

　The support capture probe is aligned immobilized, it is possible to use the same support that is used in known microarray and microarray or the like, for example, a glass slide or film, the use of such beads it can. Are described in Japanese Patent No. 4244788, etc., may be used a support shape having a plurality of protrusions on the surface. The material of the support is not particularly limited, glass, ceramics, inorganic materials such as silicon; can be exemplified polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and the like polymers such as silicone rubber.

[0033]

　As a method for immobilizing the capture probe to the support, a method of synthesizing oligo DNA on the support surface, there is known a method of dropwise oligo DNA which had been previously synthesized to a support surface fixed.

[0034]

　As the former method, Ronald et al. (U.S. Pat. No. 5,705,610), Michel et al. (U.S. Pat. No. 6,142,266), and Francesco et al. (U.S. Pat. No. 7,037,659) is . Since an organic solvent is used during the DNA synthesis reaction in these methods, it is desirable that the support is a material which is resistant to organic solvents. Further, in the Francesco et al. Method, for controlling the DNA synthesis from the back surface of the support is irradiated with light, the support is preferably a material having a light transmitting property.

[0035]

　As the latter method, mention may be made of Hirota et al. Method of using (Patent No. 3,922,454) or spotter. As a method of spot, pin system by mechanical contact of the pin tip to the solid phase, an ink jet method using the principle of an ink jet printer, capillary method and the like by capillary. After spotting process, cross-linking by UV radiation, post-processing such as blocking of the surface is carried out if necessary. For immobilizing oligo DNA covalently bonded to the surface-treated substrate surface, the end of the oligo DNA is introduced functional group such as amino group or SH group. Surface modification of the support is usually carried out by treatment with a silane coupling agent having an amino group.

[0036]

　Hybridization of each miRNA capture probe which is immobilized on a support, the extracted RNA from the specimen, a labeled nucleic acid sample with a labeling substance (the analyte nucleic acids from a sample) is prepared, the probe of the labeled nucleic acid sample carried out by contacting with. The "analyte nucleic acid from a sample", in addition to the RNA extracted from the specimen, cDNA and cRNA prepared by reverse transcription reaction from the RNA and the like. The nucleic acid sample derived from the labeled analyte may be one labeled analyte RNA directly or indirectly labeled substance, also, a cDNA or cRNA prepared from the sample RNA directly or indirectly labeled substance it may be the one that was labeled.

[0037]

　The method of binding a labeling substance to the nucleic acid sample from the sample, taken up 'method of binding a labeling substance to the end, 5' 3 of the nucleic acid sample method of binding a labeling substance to the end, a nucleotide labeled substance is bound to the nucleic acid the method can be cited to. 3 'method of binding the terminal labeling substance, 5' in the method of binding the terminal a labeling substance may be an enzyme reaction. The enzymatic reaction may be used T4 RNA Ligase and Terminal Deoxitidil Transferase, and Poly A polymerase. Any of the labeling method also "Shao-Yao Ying ed, miRNA experimental protocol, YODOSHA, 2008," the methods that are described in can be referred to. Also, a kit for binding directly or indirectly labeled substance to the end of the RNA are various commercial. For example, 3 as a 'kit to bind directly or indirectly labeled substance to the end, "3D-Gene" miRNA labeling kit (Toray), miRCURY miRNA HyPower labeling kit (Ekishikon Co.), NCode miRNA Labeling system (Life Technologies Inc.) can be exemplified FlashTag Biotin RNA Labeling Kit (Genis Sphere Inc.).

[0038]

　In addition, as in the conventional method, by synthesizing the cDNA or cRNA from the specimen RNA in the presence of the labeled deoxyribonucleotide or labeled ribonucleotide, a cDNA or cRNA labeled substance was incorporated was prepared, which array to probe hybridized with the above, the method can also be called.

[0039]

　In the present invention, the labeling substance can be used, mention may be made of various labeling substances it is also used in the known microarray analysis. Specifically, fluorescent dyes, phosphorescent dyes, enzymes, radioisotopes, and the like, without limitation. Preferred are measurements simple, it is easily detected fluorescent dye. Specifically, cyanine (Cyanine 2), aminomethyl coumarin, fluorescein, indocarbocyanine (Cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine Red, Texas Red, indocarbocyanine (Cyanine 5), cyanine 5.5, cyanine 7, but include known fluorescent dyes such as oysters, but are not limited to.

[0040]

　As the labeling substance may be a semiconductor fine particles having a light-emitting properties. Such semiconductor microparticles, such as cadmium selenide (CdSe), cadmium telluride (CdTe), indium gallium phosphide (InGaP), and the like silver indium zinc sulfide (AgInZnS).

[0041]

　As described above by contacting a nucleic acid sample from a labeled analyte and miRNA capture probe on the support, thereby hybridizing the nucleic acid sample and a probe. The hybridization step can be performed in exactly the same manner as conventional. The reaction temperature and time may be appropriately selected depending on the chain length of the nucleic acids being hybridized, in the case of nucleic acid hybridization is usually 1 minute to ten hours at about 30 ° C. - 70 ° C.. It performs hybridization, after washing, detecting the signal intensity from the label material in the individual probe immobilization region on a support. Detection of the signal intensity is performed using a suitable signal reader according to the type of labeling substance. In the case of using the fluorescent dye as a labeling substance may be used a fluorescence microscope and fluorescence scanner or the like.

[0042]

　Measurements of the detected fluorescence intensity is compared with ambient noise. Specifically, the measurements obtained from the probe immobilization region, comparing the measurements obtained from other positions, and detected a case where the former numerical values ​​are exceeded (valid determination positive) .

[0043]

　The detected measurement values, if included background noise may be subtracted background noise. The ambient noise as the background noise can be subtracted from the detected measured values. Other, "FujiFukashiKo, Katsuhisa Horimoto ed., Microarray data statistical analysis protocols, YODOSHA, 2008" may be used the method described in.

[0044]

　In the present invention, as compared process described below the measured value of the abundance of the target miRNA and reference miRNA obtained in the measuring step, may be used in the determination step, for example, the target miRNA in the body fluid sample when performing expression analysis performs a correction of the measured values of the measurements and the reference miRNA target miRNA, a measurement value after correction and the expression level may be carried out comparing step and judging step by using the expression level . That is, the measurement step may include the process of correcting the measurement value of the target miRNA and reference miRNA.

[0045]

　As the correction method, conventional methods can be used, for example, a global normalization method of performing correction using the measured total miRNA detected, and a quantile normalization method. Further, U1 snoRNA, U2 snoRNA, U3 snoRNA, U4 snoRNA, U5 snoRNA, U6 snoRNA, 5S rRNA, housekeeping RNA such 5.8S rRNA (JP 2007-75095 JP; see, JP 2007-97429 JP) Ya , specific correction endogenous miRNA (Roberts, TC et al., 2014, PLoS ONE, 9 (2) winding, e89237; Chen, X. et al., 2013, PLoS ONE, 8 (11) winding, E79652; it may be corrected using the WO see, 2016/043170), may be corrected by using an external standard nucleic acid added at the time of RNA extraction and labeling. "Endogenous" artificial rather than being added to the sample, which means that naturally present in the sample. For example, if said "endogenous miRNA" refers to a miRNA derived from that naturally present in the sample, it provided the sample organism. If applying the method of the present invention in the expression analysis of a target miRNA in the body fluid sample the quality evaluation of the miRNA, it is preferable to use a correction method using the external standard nucleic acid, such as spike control that is not dependent on the sample.

[0046]

　comparison step of the present invention, the abundance measurements or representative values of one or more reference miRNA in body fluids samples obtained in the measuring step, one or more reference miRNA in standard bodily fluid analyte the amount of presence measurements or as compared to the representative value is a step of obtaining a difference or ratio of the abundance measurements or the representative value of each reference miRNA in both specimens. Here, the difference or ratio of the abundance measurements or the representative value of the reference miRNA typically refers to the difference or ratio determined by the following equation.
Difference in abundance measurements or the representative value of the reference miRNA
= (abundance measurements or representative values in a body fluid sample) - (abundance measurements or representative value in the standard bodily fluid analyte) (Formula I)
reference the ratio of the abundance measurements or the representative value of the miRNA
= (abundance measurements or representative values in a body fluid sample) / (abundance measurements or representative value in the standard bodily fluid analyte) (formula II)

[0047]

　However, the formula I, instead of Formula II, Formula I order the following that the reverse calculation ', the formula II' no problem even if calculating the difference or ratio in.
Difference in abundance measurements or the representative value of the reference miRNA
= (abundance measurements or representative value in the standard bodily fluid analyte) - (abundance measurements or representative values in a body fluid sample) (formula I ')
the ratio of the abundance measurements or the representative value of the reference miRNA
= (abundance measurements or representative value in the standard bodily fluid analyte) / (abundance measurements or representative values in a body fluid sample) (formula II ')

[0048]

　The difference or ratio of abundance measurements or representative value of the reference miRNA, the difference or may be converted to the logarithm after calculating the ratio, calculates a difference or ratio previously abundance in the sample from the logarithmic conversion it may be. In the present invention, the logarithmic value, bottom means a value converted to 2 logarithm.

[0049]

　As described later in the description of the determination step, of the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, in the case of using a single miRNA based miRNA is abundance measurements of the reference miRNA contained in the body fluid sample it may be used to determine seeking difference or ratio of the abundance measurements of the reference miRNA in the value and the standard bodily fluid analyte. In the case of using a plurality of miRNA based miRNA is the abundance measurements of the plurality of reference miRNA included in the representative value and the standard bodily fluid analyte abundance measurements of the plurality of reference miRNA contained in the body fluid sample It obtains a representative value can be used to determine seeking the difference or ratio of the two representative values. Representative values, as described below, can be used an average or median. Alternatively, the Samata the abundance measurements in abundance measurements and the standard bodily fluid analyte in a body fluid sample for each individual reference miRNA determine the specific, it is determined in the next decision step, according to predetermined criteria for each reference miRNA , it is possible to determine the quality of the quality of miRNA contained in the body fluid sample.

[0050]

　determining step of the present invention, the abundance measurements or representative value of 1 or more criteria miRNA in a body fluid sample obtained in the comparing step, one or more reference miRNA in the standard bodily fluid analyte the Samata the abundance measurements or the representative value based on the ratio, a step of determining acceptability of the quality of miRNA contained in the body fluid sample. quality determination of the quality of the miRNA, for difference or ratio of 1 or abundance measurements of a plurality of reference miRNA or a representative value contained in the body fluid sample and the standard bodily fluid analyte, the criterion for determining the quality of the quality setting a threshold in advance, it is possible to determine the quality (good or bad) quality depending on whether exceeds the threshold value. That is, the difference or ratio of abundance measurements or the representative value of the reference miRNA obtained by the above formula I or formula II, if it exceeds a pre arbitrarily determined threshold, the good quality of the miRNA contained in the body fluid sample It determines that, if the difference or ratio of the abundance measurements or the representative value of the reference miRNA is the threshold value or less, the quality of miRNA contained in the body fluid sample can be determined to be defective. Alternatively, when a difference or ratio obtained by the formula I 'or formula II', when below a threshold as a reference to which the difference or ratio is predetermined arbitrarily, the quality of miRNA contained in the body fluid sample was determined to be good, if the difference or ratio is equal to or greater than the threshold value, the quality of the miRNA contained in the body fluid sample can be determined to be defective.

[0051]

　In the determination step, as described above, the difference or ratio of the abundance measurements or the representative value of each reference miRNA obtained in Comparative process is converted into the logarithmic, determination may be performed using the logarithmic value.

[0052]

　Of the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, in the case of using a single miRNA based miRNA, in comparison step, the abundance measurements of the reference miRNA and the standard bodily fluid analyte contained in a body fluid sample obtaining a difference or ratio of the abundance measurements of the reference miRNA included, whether or not the value of the difference or ratio exceeds a threshold value as a reference (in the case of formula I, II), or whether below (formula I ', II' case) makes it possible to determine the quality of the quality.

[0053]

　Based miRNA, in the case of using a plurality of reference miRNA of miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, a representative value and the standard fluid of abundance measurements of the plurality of reference miRNA contained in the body fluid sample obtains a representative value of abundance measurements of the plurality of reference miRNA contained in the sample, whether these representative values ​​of the differences or whether more than a threshold ratio as a reference (in the case of formula I, II), or below or by (in the case of formula I ', II') can be judged good or bad quality. Here, as the representative value, it is possible to use the average or median abundance of a plurality of reference miRNA.

[0054]

　With reference to miRNA, in the case of using a plurality of reference miRNA of miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, abundance measurements and the standard bodily fluid analyte contained in a body fluid sample for each individual reference miRNA Samata the abundance measurement values ​​included in the calculated ratio, whether more than whether / below a threshold value as a reference for each reference miRNA may determine. In this case, such as by the prioritization and weighting to each of the determination by the plurality of reference miRNA, it is preferable to provide additional criteria. When using one reference miRNA is may be arbitrarily selecting one miRNA from miRNA shown in SEQ ID NO: 1-12, can be selected that amount present depending on the storage time is significantly reduced preferably, for example, hsa-miR-125a-3p (SEQ ID NO: 1), it is preferable to select one of hsa-miR-125b-1-3p (SEQ ID NO: 2). Further, when the evaluation of the more stringent or high accuracy, it is preferable to use a plurality of reference miRNA. For example, more preferred to use 2 to 5 standard miRNA, in particular, it is preferable to select two hsa-miR-125a-3p and hsa-miR-125b-1-3p. However, as described in the Examples below, even when adopting either the hsa-miR-125a-3p and hsa-miR-125b-1-3p only as one of a plurality of reference miRNA, sufficiently precisely it is possible to determine the quality of the quality. Further, for example, a case for the purpose of gene expression analysis, if the target miRNA is expressed analyzed corresponds to one of the miRNA of SEQ ID NO: 1 to 12, the reference miRNA from miRNA excluding the target miRNA it may be selected.

[0055]

　Threshold as a reference for determination may be set arbitrarily according to the purpose and determining the accuracy of the evaluation. For example, it is possible to set the abundance measurements of the reference miRNA contained in the standard bodily fluid analyte threshold.

[0056]

　Hereinafter, the determination step in the case of calculating the difference or ratio of the abundance measurements or representative value of the reference miRNA according Formula I or Formula II, be described more specifically. If according to formula I 'or formula II' is prepared analogously as described below employing the appropriate threshold, it may determine the quality of the miRNA and good when below its threshold.

[0057]

　When using one reference miRNA, for example, according to the judgment criteria as shown in formula 1A ~ 9A or Formula 1B ~ 9B, the presence of the reference miRNA in abundance measurements and the standard bodily fluid analyte reference miRNA in bodily fluid analyte and comparing the amount measured values, it is possible to determine the quality of the quality.

[0058]

　As shown in Equation 1A, the ratio of abundance measurements E of the reference miRNA in abundance measurements e and the standard bodily fluid analyte reference miRNA in a body fluid specimen (e / E) calculated, this value exceeds the threshold value t1 the quality of the miRNA contained in the body fluid sample can be determined that good when. Threshold t1 is preferably 0.7 or more, more preferably 0.8 or more.
　　e / E> t1 (Equation 1A)

[0059]

　Further, as shown in equation 2A, the difference in abundance measurements E of the reference miRNA in abundance measurements e and the standard bodily fluid analyte reference miRNA in a body fluid specimen (e-E) calculated, the value is the threshold value t2 it is possible to determine the quality of the miRNA contained in the body fluid sample with good when exceeding. Since its abundance on the type of miRNA may be different, the threshold t2 can be arbitrarily set according to the reference miRNA used for the determination. For example, the threshold value t2 can be set in the range of -50 or 0 or less, when determined by the more stringent criteria can be set in the range of -20 or 0 or less. For example, the threshold t2 is set to 0, if the difference of abundance measurements (e-E) is greater than zero (positive), it is possible to determine the quality of the miRNA contained in the body fluid sample with good.
　　e-E> t2 (Equation 2A)

[0060]

　Also, it may be employed abundance measurements E of the reference miRNA in standard bodily fluid analyte as a threshold t3, that case, as shown in Equation 3A, the threshold abundance measurements e criteria miRNA in bodily fluid analyte t3, that is, to determine if the excess of abundance measurements E of the reference miRNA in standard bodily fluid analyte, the quality of miRNA contained in the body fluid sample with good. This corresponds to the case where the threshold t2 and 0 in formula 2A. Therefore, Equation 3A corresponds to one embodiment of Formula 2A. That is, the formula 3A corresponds to the determination based on the difference of abundance measurements.
　　e> E (= t3) (formula 3A)

[0061]

　In addition to degradation of miRNA in a body fluid sample, experimental operation, measurement when considering factors to affect results, endogenous miRNA is a miRNA that is stable that do not depend on the degradation of RNA (hereinafter, "non-degraded endogenous miRNA "that.) may be determined using. Nondegradable endogenous miRNA is in the body fluid sample is a miRNA that contains a certain amount without depending on the type, etc. preferably those immediately obtained or prepared prior to RNA degradation (analyte contained therein when the degradation of the nucleic acid sample after availability or preparation of the time) as not progressing after RNA degradation (sample a predetermined time has elapsed, is expected to degradation of the nucleic acid sample is proceeding contained therein ) ratio of abundance measurements with 0.90 or more, more preferably it is possible to select a miRNA such that less than 0.95. For example, it is possible to use and hsa-miR-149-3p having the nucleotide sequence represented by SEQ ID NO: 25, etc. hsa-miR-4463 having the nucleotide sequence represented by SEQ ID NO: 26 as a non-degradable endogenous miRNA. When performing expression analysis of a target miRNA in the body fluid sample is a "correction endogenous miRNA" as used in the above correction processing can be commonly used as a "non-degradable endogenous miRNA."

[0062]

　When determining the quality of the quality of miRNA contained in the body fluid sample by utilizing a non-degradable endogenous miRNA, for example, as shown in Equation 4A, abundance measurements e and non-degraded reference miRNA in bodily fluid analyte the ratio of the abundance measurements c of endogenous miRNA (abundance ratio e / c), and, with the abundance Found C of the abundance measurements E of the reference miRNA in standard bodily fluid analyte nondegradable endogenous miRNA determine the specific (abundance ratio E / C), when the ratio of these two respective abundance ratios exceeds the threshold value t4, it is possible to determine the quality of the miRNA contained in the body fluid sample with good.
　Alternatively, as shown in Equation 5A, the difference between the abundance measurements c abundance measurements e and non-degradable endogenous miRNA reference miRNA in a body fluid sample (abundance difference e-c), and, standard bodily fluid analyte obtains the difference (abundance difference E-C) of the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in the ratio of these two respective abundance difference exceeds the threshold value t5 If, it is possible to determine the quality of the miRNA contained in the body fluid sample with good. Threshold t4 and t5 in this case is preferably 0.7, more preferably 0.8.
　Formula 4A, wherein 5A corresponds to the judgment based on the ratio of abundance measurements.
　　(E / c) / (E / C)> t4 ( formula 4A)
　　(e-c) / (E-C)> t5 (Formula 5A)

[0063]

　Further, as shown in Equation 6A, the ratio of the abundance measurements c abundance measurements e and non-degradable endogenous miRNA reference miRNA in a body fluid sample (abundance ratio e / c), and, standard bodily fluid analyte determine the specific (abundance ratio E / C) of the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in the difference between these two respective abundance ratios exceeds the threshold value t6 If, it is possible to determine the quality of the miRNA contained in the body fluid sample with good.
　Alternatively, as shown in Equation 7A, the difference between the abundance measurements c abundance measurements e and non-degradable endogenous miRNA reference miRNA in a body fluid sample (abundance difference e-c), and, standard bodily fluid analyte the difference (the abundance difference E-C) determined the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in the difference between these two respective abundance difference exceeds the threshold value t7 If, it is possible to determine the quality of the miRNA contained in the body fluid sample with good. Threshold t6 and t7, for example can be set in a range of -50 or 0 or less, may be, for example, 0.
　Formula 6A, wherein 7A corresponds to the determination based on the difference of abundance measurements.
　　(E / c) - (E / C)> t6 ( formula 6A)
　　(e-c) - (E-C)> t7 (Equation 7A)

[0064]

　Further, as the threshold t8, may employ the ratio of the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in standard bodily fluid analyte (abundance ratio E / C), its If, as shown in equation 8A, the ratio (abundance ratio e / c) threshold t8 of the abundance measurements e criteria miRNA and abundance measurements c of non-degradable endogenous miRNA in a body fluid sample, i.e. when exceeding the ratio of the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in standard bodily fluid analyte (abundance ratio E / C), the quality of the miRNA contained in the body fluid sample it can be determined that good. This is because, in case of employing the equation 6A, corresponds to a case where the threshold value t6 and 0. Therefore, Equation 8A is an embodiment of Formula 6A, corresponds to a determination based on the difference in the abundance measurements.
　Alternatively, as the threshold t9, may employ the difference (abundance difference E-C) of the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in standard bodily fluid analyte, the If, as shown in equation 9A, the difference (abundance difference e-c) threshold t9 of the abundance measurements e criteria miRNA and abundance measurements c of non-degradable endogenous miRNA in a body fluid sample, i.e. If the above difference (abundance difference E-C) of the abundance Found C abundance measurements E and non-degradable endogenous miRNA reference miRNA in standard bodily fluid analyte, the quality of miRNA contained in the body fluid sample it can be determined that good. This is because, in case of employing the equation 7A, which corresponds to a case where the threshold t7 zero. Therefore, Equation 9A is an embodiment of Formula 7A, corresponding to the determination based on the difference of abundance measurements.
　　e / c> E / C ( = t8) ( Equation 8A)
　　e-c> E-C (= t9) (Formula 9A)

[0065]

　As in the case of using the reference miRNA multiple miRNA, calculated representative value of the abundance measurements of a plurality of reference miRNA in a body fluid sample, a representative value of abundance measurements of the plurality of reference miRNA in standard bodily fluid analyte , it can be used to determine seeking the difference or ratio of the two representative values. Specifically, in the judgment criteria shown in the above formulas 1A ~ formula 9A, a representative value r abundance measurements of a plurality of reference miRNA in bodily fluid analyte in place of the abundance measurements e criteria miRNA in bodily fluid analyte , a representative value R of the abundance measurements of a plurality of reference miRNA in standard bodily fluid analyte in place of the abundance measurements E of the reference miRNA in standard bodily fluid analyte, may be used, respectively. That may be determined using either equation 1B ~ formula 9B below. Representative values can be used an average or median of the measurement values.
　　r / R> t1 (Equation 1B)
　　r-R> t2 (Equation 2B)
　　r> R (= t3) (Formula 3B)
　　(r / c) / (R / C)> t4 (formula 4B)
　　(r-c ) / (R-C)> t5 ( formula 5B)
　　(r / c) - (R / C)> t6 (equation 6B)
　　(r-c) - (R-C)> t7 (equation 7B)
　　r / c > R / C (= t8) ( equation 8B)
　　r-c> R-C (= t9) (formula 9B).

[0066]

　Here, the above formulas 1A-formula 9A, in the formula 1B-type 9B, in consideration of the error of the experiments, to have a width of certain errors alpha threshold t1 ~ t9, respectively "t1 ± alpha" - it may be used as the "t9 ± α". This error α where may be arbitrarily set, but for example, in the formula 2A can be made to have a width of about 10% to the threshold t2 is set as α in E.

[0067]

　Further, as the threshold values ​​may be used after converting the measured values ​​of the abundance in logarithm. In this case, it is sufficient to set an appropriate threshold to match that conversion. For example, when applying the formula 1A, and converts the abundance ratio of the reference miRNA the (e / E) to the logarithm may be set threshold t1 together on the conversion. In this case, consequently, abundance measurements e, thereby obtaining the difference between the logarithm of E.

[0068]

　Further, Samata obtains the ratio of the abundance measurements of abundance measurements and in standard bodily fluid analyte in a body fluid sample for each individual reference miRNA, a judgment according to the judgment criteria for each reference miRNA, comprehensively and the results quality of the quality of the miRNA contained in the body fluid sample Te can be determined.

[0069]

　Specifically, for example, in the determination for each criterion miRNA, when the number of reference miRNA that has been determined as good is greater than a predetermined number of number or any of the determined reference miRNA defective, miRNA contained in the body fluid sample it is possible to determine the quality of the good. Conversely, the number of the determined reference miRNA and failure, when above a number or a predetermined number of reference miRNA that has been determined to be good, it is possible to determine the quality of the miRNA to be included in the sample and failure. Further, when the evaluation of the more stringent or high accuracy, priority may be given a bad decision by a particular one of the reference miRNA than the number of reference miRNA gave good judgment. That is, such a case the determination result of the particular one of the reference miRNA is poor, regardless of the number of reference miRNA of good judgment, the quality of the miRNA may be determined to be defective contained in the body fluid sample. Such particular one reference miRNA, can be preferably used any of hsa-miR-125a-3p (SEQ ID NO: 1) and hsa-miR-125b-1-3p (SEQ ID NO: 2).

[0070]

　The present invention, in accordance with miRNA quality evaluation method of the present invention, in order to evaluate the quality of the miRNA from bodily fluid analyte, one or more computers,
　were prepared from the body fluid sample and standard bodily fluid analyte, including miRNA were measured respectively using the RNA samples to obtain the abundance measurements in body fluid sample and standard bodily fluid analyte of one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, measured value acquisition step;
　an abundance measurements or representative values of one or more reference miRNA in bodily fluid analyte, compared to the abundance measurements or representative value of 1 or more criteria miRNA in standard bodily fluid analyte to obtain a difference or ratio of the abundance measurements or representative value of 1 or more criteria miRNA between bodily fluid analyte and standard bodily fluid analyte, comparing step; and
　obtained in the comparing step, one or more reference miRNA abundance measurements or Based on the difference or ratio of the representative values, determining the acceptability of the quality of the miRNA from bodily fluid analyte, determining step
for executing (i.e., includes instructions to execute the above steps in one or more computers) program, and a computer-readable recording medium recording the program.

[0071]

　For example, the program is incorporated in the device for analyzing the expression level of miRNA, the measured value acquisition step, the reference miRNA expression levels separate expression measurement apparatus was measured and the expression measurement unit or the device of the apparatus (i.e. in the sample the acquired measurements of the reference miRNA abundance), each step may be implemented using a measured value. Measurements to be acquired, may be corrected measurements. Further, the program may also include instructions for executing the processing for correcting the measured value acquired in a computer. Details of each step are as described above with respect to miRNA quality evaluation method of the present invention.

[0072]

　The "program" is a data processing method written in any computer language and written method, may be of any format such as source code or binary code. Note that "program" is not necessarily limited to those composed singularly, what is distributed configuration as a plurality of modules or libraries, or in cooperation with a different program such as the OS (Operating System) that also including those to achieve the function. Specific configuration for reading a recording medium, reading procedure or, for such installation procedure after reading, may be well-known configuration and procedure.

[0073]

　"Recording medium", a flexible disk, a magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, may be any "fixed physical medium" such as a DVD (non-transitory recording medium). Alternatively, LAN, WAN, typified by the Internet, such as a communication line or a carrier wave when over the network to transmit the program, may be a short-term holding the program "communications media."

[0074]

　The present invention also provides a probe for capturing one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1 to 12 comprising the immobilized support, the miRNA quality evaluation chip provide. Further, the present invention includes a probe for capturing a target miRNA, a probe for capturing one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12 is immobilized comprising a support, to provide a chip miRNA expression analysis. Here, the target miRNA, 1 or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12, a probe for capturing these also support these capture probes are immobilized body is as described above.

[0075]

　miRNA expression analysis chip of the present invention, the housekeeping RNA used in the correction process, the specific correction endogenous miRNA, correction nucleic acid, such as an external standard nucleic acid to be added, particularly for capturing the correction endogenous miRNA probes may be further immobilized on a support.

[0076]

　In the present invention, miR-125a-3p, miR-125b-1-3p, miR-3184-5p, miR-4443, miR-4638-5p, miR-4746-3p, miR-5572, miR-575, miR -6798-5p selection, miR-7110-5p, miR-887-3p, and one or more miRNA selected from miR-939-5p, from preferably consisting of the nucleotide sequence represented by SEQ ID NO: 1 ~ 12 miRNA one or more miRNA is used as a reference miRNA for measuring the degradation of body fluid sample derived RNA. If the body fluid sample is a body fluid sample of the human, one or more miRNA is selected from the miRNA comprising the nucleotide sequence shown in SEQ ID NO: 1-12 may be used as a reference miRNA.

[0077]

　The term "miR-125a-3p gene" or "miR-125a-3p" is human miR-125a-3p (i.e. hsa-miR-125a-3p, miRBase Accession No. MIMAT0004602) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 1 is the sequence of hsa-miR-125a-3p. hsa-miR-125a-3p genes, Lagos-Quintana M et al., 2002, Curr Biol, 12 vol, may be obtained by the method described in P735-739. In addition, the term "miR-125a-3p", the precursor to take a hairpin-like structure "mir-125a" is also included, for example, the word "hsa-miR-125a-3p" hsa-mir-125a (miRBase Accession No. MI0000469, SEQ ID NO: 13) are also included.

[0078]

　"MiR-125b-1-3p gene" or the term "miR-125b-1-3p" includes humans of miR-125b-1-3p (i.e. hsa-miR-125b-1-3p, miRBase Accession No. MIMAT0004592 ) and other, including such species homologs or orthologs. RNA sequence shown in SEQ ID NO: 2 is the sequence of hsa-miR-125b-1-3p. hsa-miR-125b-1-3p gene, Lagos-Quintana M et al., 2002, Curr Biol, 12 vol, may be obtained by the method described in P735-739. Also, the term "miR-125b-1-3p", its precursor taking a hairpin-like structure "mir-125b-1" is also encompassed, for example the word "hsa-miR-125b-1-3p" the hsa-mir-125b-1 (miRBase Accession No. MI0000446, SEQ ID NO: 14) are also included.

[0079]

　The term "miR-3184-5p gene" or "miR-3184-5p" is the human miR-3184-5p (i.e. hsa-miR-3184-5p, miRBase Accession No. MIMAT0015064) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 3 is the sequence of hsa-miR-3184-5p. hsa-miR-3184-5p gene, Stark MS et al., 2010, PLoS One, 5 vol, may be obtained by the method described in E9685. In addition, "miR-3184-5p" The word is, the precursor to take a hairpin-like structure "mir-3184" is also included, for example, "hsa-miR-3184-5p" is the word that hsa-mir-3184 (miRBase Accession No. MI0014226, SEQ ID NO: 15) are also included.

[0080]

　The term "miR-4443 gene" or "miR-4443" encompasses such human miR-4443 (i.e. hsa-miR-4443, miRBase Accession No. MIMAT0018961) and other species homologs or orthologs. RNA sequence shown in SEQ ID NO: 4 is the sequence of hsa-miR-4443. hsa-miR-4443 gene, Jima DD et al., 2010, Blood, 116 vol, may be obtained by the method described in P118-127. Further, "miR-4443" The term, its precursors take a hairpin-like structure "mir-4443" are encompassed, for example, "hsa-miR-4443" in terms hsa-mir-4443 (miRBase Accession No . MI0016786, SEQ ID NO: 16) are also included.

[0081]

　The term "miR-4638-5p gene" or "miR-4638-5p" is the human miR-4638-5p (i.e. hsa-miR-4638-5p, miRBase Accession No. MIMAT0019695) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 5 is the sequence of hsa-miR-4638-5p. hsa-miR-4638-5p gene can be obtained Persson H, et al., 2011, Cancer Res, 71 vol., by the method described in P78-86. In addition, "miR-4638-5p" The word is, the precursor to take a hairpin-like structure "mir-4638" is also included, for example, "hsa-miR-4638-5p" is the word that hsa-miR-4638 (miRBase Accession No. MI0017265, SEQ ID NO: 17) are also included.

[0082]

　The term "miR-4746-3p gene" or "miR-4746-3p" is the human miR-4746-3p (i.e. hsa-miR-4746-3p, miRBase Accession No. MIMAT0019881) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 6 is the sequence of hsa-miR-4746-3p. hsa-miR-4746-3p gene can be obtained Persson H, et al., 2011, Cancer Res, 71 vol., by the method described in P78-86. In addition, "miR-4746-3p" The word is, the precursor to take a hairpin-like structure "mir-4746" is also included, for example, "hsa-miR-4746-3p" is the word that hsa-mir-4746 (miRBase Accession No. MI0017385, SEQ ID NO: 18) are also included.

[0083]

　The term "miR-5572 gene" or "miR-5572" encompasses such human miR-5572 (i.e. hsa-miR-5572, miRBase Accession No. MIMAT0022260) and other species homologs or orthologs. RNA sequence shown in SEQ ID NO: 7 is the sequence of hsa-miR-5572. hsa-miR-5572 gene can be obtained Tandon M et al., 2012, Oral Dis, 18 vol., by the method described in P127-131. Further, "miR-5572" The term, its precursors take a hairpin-like structure "mir-5572" are encompassed, for example, "hsa-miR-5572" in terms hsa-mir-5572 (miRBase Accession No . MI0019117, SEQ ID NO: 19) are also included.

[0084]

　The term "miR-575 gene" or "miR-575" encompasses such human miR-575 (i.e. hsa-miR-575, miRBase Accession No. MIMAT0003240) and other species homologs or orthologs. RNA sequence shown in SEQ ID NO: 8 is the sequence of hsa-miR-575. hsa-miR-575 gene, Cummins JM et al., 2006, Proc Natl Acad Sci USA, 103 vol, may be obtained by the method described in P3687-3692. Also, the term "miR-575", its precursor taking a hairpin-like structure "mir-575" are also encompassed, for example, "hsa-miR-575" is the term hsa-mir-575 (miRBase Accession No . MI0003582, SEQ ID NO: 20) are also included.

[0085]

　The term "miR-6798-5p gene" or "miR-6798-5p" is the human miR-6798-5p (i.e. hsa-miR-6798-5p, miRBase Accession No. MIMAT0027496) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 9 is the sequence of hsa-miR-6798-5p. hsa-miR-6798-5p gene can be obtained Ladewig E et al, 2012, Genome Res, 22 vol., by the method described in P1634-1645. In addition, "miR-6798-5p" The word is, the precursor to take a hairpin-like structure "mir-6798" is also included, for example, "hsa-miR-6798-5p" is the word that hsa-mir-6798 (miRBase Accession No. MI0022643, SEQ ID NO: 21) are also included.

[0086]

　The term "miR-7110-5p gene" or "miR-7110-5p" is the human miR-7110-5p (i.e. hsa-miR-7110-5p, miRBase Accession No. MIMAT0028117) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 10 is the sequence of hsa-miR-7110-5p. hsa-miR-7110-5p gene can be obtained Ladewig E et al, 2012, Genome Res, 22 vol., by the method described in P1634-1645. In addition, "miR-7110-5p" The word is, the precursor to take a hairpin-like structure "mir-7110" is also included, for example, "hsa-miR-7110-5p" is the word that hsa-mir-7110 (miRBase Accession No. MI0022961, SEQ ID NO: 22) are also included.

[0087]

　The term "miR-887-3p gene" or "miR-887-3p" is the human miR-887-3 (i.e. hsa-miR-887-3p, miRBase Accession No .MIMAT0004951) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 11 is the sequence of hsa-miR-887-3p. hsa-miR-887-3p gene can be obtained Berezikov E et al, 2006, Genome Res, 16 vol., by the method described in P1289-1298. In addition, "miR-887-3p" The word is, the precursor to take a hairpin-like structure "mir-887" is also included, for example, the word "hsa-miR-887-3p" hsa-mir-887 (miRBase Accession No. MI0005562, SEQ ID NO: 23) are also included.

[0088]

　The term "miR-939-5p gene" or "miR-939-5p" is the human miR-939-5p (i.e. hsa-miR-939-5p, miRBase Accession No. MIMAT0004982) and other species homolog or It encompasses such as orthologs. RNA sequence shown in SEQ ID NO: 12 is the sequence of hsa-miR-939-5p. hsa-miR-939-5p gene, Lui WO, et al., 2007, Cancer Res, 67 vol, may be obtained by the method described in P6031-6043. Also, the term "miR-939-5p", that takes a hairpin-like structure as a precursor "mir-939" are encompassed, hsa-miR in terms such as "hsa-miR-939-5p" 939 (miRBase Accession No. MI0005761, SEQ ID NO: 24) are also included.
Example

[0089]

　Hereinafter, including the process that selects the reference miRNA that depends on the degradation of RNA, now it is described by way of the present invention embodiment. However, the present invention is not limited to the following examples.

[0090]

Selection of reference miRNA
(DNA microarrays)
　the following experiment was conducted using a Toray Co., Ltd. "3D-Gene" human miRNA oligo chip (miRBase release 21 corresponds).

[0091]

(Preparation of serum samples)
　was collected respectively from a healthy person three, and sera were prepared. The resulting serum was dispensed to six per 300μL one by one person, it was allowed to stand in a refrigerator set five out to 4 ℃. One per person was housed in a freezer set to immediately -80 ℃ (0 hours). For serum samples and left in the refrigerator for 6 hours, 24 hours, 48 hours and collected respectively after 72 hours and 168 hours, it was housed in a freezer set at -80 ° C.. Specimens accommodated in -80 ° C. freezer until RNA extraction procedure described below, was allowed to stand.

[0092]

(Sample measurement of RNA Preparation and miRNA expression levels)
　are prepared as described above, were simultaneously dissolved stand serum of the freezer, RNA included in the serum samples were extracted (hereinafter, referred to as sample RNA.). The extraction, "3D-Gene" RNA extraction reagent from liquid sample kit ( Toray Industries, Inc.) was used.

[0093]

　The resulting sample RNA, were labeled with "3D-Gene" miRNA labeling kit (Toray). During labeling, in order to correct the measured value of the miRNA expression level, it was added an external standard nucleic acid. For labeled specimen RNA, using the "3D-Gene" miRNA chip (Toray Industries, Inc.), in accordance with the standard protocol, hybridization was performed. DNA microarrays after hybridization and fluorescence intensity was measured and subjected to microarray scanner (Toray). Scanner settings, the laser output of 100%, the voltage setting of the photomultiplier was AUTO setting.

[0094]

　The measurement for each miRNA detected by DNA microarray, the bottom is converted to 2 logarithm, performs correction by external standard nucleic acid added during labeling was obtained the expression level of each miRNA.

[0095]

　As described above, 0,6,24,48,72,168 hours after the detection of a DNA microarray, the stationary serum at 4 ° C., 3 times the expression level of each miRNA when passed between the standing measured to obtain the average value.

[0096]

(Reference miRNA selection)
　comparing the miRNA expression level of each serum sample obtained as described above, the expression level depending on the standing time (the amount present in the sample) is extracted large miRNA varying degrees of that is, it was the choice of criteria miRNA.

[0097]

　First, with reference to the expression level of each miRNA when standing 0 hour, the ratio of the expression level of each miRNA when standing 6,24,48,72,168 hours elapsed ((6, 24, 48, 72 or the expression level of at 168 hours after) / 0 hours expression levels) was obtained.

[0098]

　Next, among the detected miRNA, and narrowing to a miRNA to be detected stably with high expression region.

[0099]

　From among narrowed miRNA, for selecting a miRNA extent greater expression level depending on the elapsed varies between standing, package statistical language "R", "SAM" (Tusher VG et. Al., Proc. Natl. Acad. Sci. USA, using 2001,98 (9), 5116-5121), was extracted miRNA statistics SAM is -1. Top 12 one miRNA extracted and shows the expression level ratios in Table 1.

[0100]

　miRNA shown in Table 1 (SEQ ID NO: 1-12) are referred to 4 ° C., under a condition that the miRNA in serum is stored in a relatively unstable state, over time expressed amount is decreased, the degree thereof is large It was. Further, in the expression amount of all miRNA that standing period was detected in serum samples and serum samples at 168 hours after the time 0 hours in the refrigerator, that the correlation coefficient between the two serum specimens were 0.95 or less from, the degradation of RNA in the serum samples it was confirmed that is in progress by the elapsed time 168 hours. Therefore, miRNA shown in Table 1, depending on the degradation of RNA in serum samples, over time its expression level (in the sample abundance) it has been confirmed that can be used as miRNA index varies. That, miRNA shown in Table 1, by measuring the expression level, was found to be possible to know serum specimens miRNA of quality (degree of degradation). Particularly hsa-miR-125a-3p (SEQ ID NO: 1) and hsa-miR-125b-1-3p (SEQ ID NO: 2), early shows a keen variation from (at 48 hours later), a more accurate It was found to be suitable for evaluation of quality.

[0101]

[Table 1]

[0102]

　From the results of Example 1, the following Examples 2-6, the threshold value of the expression level ratio of the reference miRNA set to 0.8, when the above which was determined the quality of the miRNA contained in the sample as good.

[0103]

　blood was collected from healthy human one person, to prepare serum samples. The resulting serum was dispensed 300μL fraction were housed in a freezer set to immediately -80 ° C.. The extraction of RNA is, "3D-Gene" RNA extraction reagent from liquid sample kit ( Toray Industries, Inc.) was used.

[0104]

　The resulting sample RNA, and labeled with "3D-Gene" miRNA labeling kit (Toray Industries, Inc.), at the time of labeling, in order to correct the measured value of the miRNA expression level, was added an external standard nucleic acid. Labeled analyte-derived RNA is used "3D-Gene" miRNA chip (Toray), in accordance with the standard protocol, hybridization was performed. DNA microarrays after hybridization and fluorescence intensity was measured and subjected to microarray scanner (Toray). Scanner settings, the laser output of 100%, the voltage setting of the photomultiplier was AUTO setting. The signal value of the detected miRNA, is corrected by signal values ​​of the external standard nucleic acid, to give the expression level.

[0105]

　Based miRNA used for the determination of the quality, was selected hsa-miR-125b-1-3p (SEQ ID NO: 2). Further, the serum samples that are commercially available, was used as the standard bodily fluid analyte is a state where the degradation of the nucleic acid sample is not in progress, in the same manner as described above, hsa-miR-125b-1- included in the standard bodily fluid analyte to obtain the expression level of 3p.

[0106]

　The expression level of hsa-miR-125b-1-3p from serum specimens, return divided by the expression level of hsa-miR-125b-1-3p from standard bodily fluid analyte was determined both in the expression level ratios. Expression level ratio of 0.99, since it exceeds the threshold value 0.8, and determine the quality of the miRNA contained in the serum sample as good.

[0107]

　On the other hand, the expression amount of all miRNA from the detected serum specimens, the correlation coefficient between the expression amount of all miRNA from standard bodily fluid analyte of 0.99, that the quality of the miRNA was not decomposed better shown It has been. This was consistent with the determination result of the quality due to the present invention.

[0108]

　based miRNA used for the determination of the quality, instead of hsa-miR-125b-1-3p (SEQ ID NO: 2), hsa-miR-125b-1-3p (SEQ ID NO: 2) and hsa-miR change in the use of two -6798-5P (SEQ ID NO: 9), other in the same manner as in example 2, was measured the expression levels of these two miRNA from serum specimens derived and standard bodily fluid analyte .

[0109]

　Comparison of expression levels of both specimens was performed and the mean value of the expression level of the two miRNA as a representative value. A representative value of serum samples divided by the representative value of the standard bodily fluid analyte, to determine the expression level ratio. As a result, the expression level ratio is 0.98, since it exceeds the threshold value 0.8, and determine the quality of the miRNA contained in the serum sample as good.

[0110]

　On the other hand, the expression amount of all miRNA from the detected serum specimens, the correlation coefficient between the expression amount of all miRNA from standard bodily fluid analyte of 0.99, that the quality of the miRNA was not decomposed better shown It has been. This was consistent with the determination result of the quality due to the present invention.

[0111]

　The serum specimens, after serum preparation were changed to the after standing 168 hours at 4 ° C., other in the same manner as in Example 3, 2 from serum samples from and standard bodily fluid analyte expression was measured quantity of seed miRNA (hsa-miR-125b- 1-3p ( SEQ ID NO: 2) and hsa-miR-6798-5p (SEQ ID NO: 9)). The average value of the expression level of the two miRNA as a representative value, to determine the expression level ratio from the representative value.

[0112]

　As a result, the expression level ratio is 0.34, since it falls below the threshold value 0.8, the quality of miRNA contained in the serum sample was determined to be defective.

[0113]

　On the other hand, the expression amount of all miRNA from the detected serum specimens, a low value of 0.93 correlation coefficient between the expression amount of all miRNA from standard bodily fluid analyte, occurs decomposition of miRNA, quality has deteriorated was it was shown. This was consistent with the determination result of the quality due to the present invention.

[0114]

　serum specimens, after serum preparation were changed to the after standing 72 hours at 4 ° C., other in the same manner as in Example 3, 2 from serum samples from and standard bodily fluid analyte expression was measured quantity of seed miRNA (hsa-miR-125b- 1-3p ( SEQ ID NO: 2) and hsa-miR-6798-5p (SEQ ID NO: 9)). determined hsa-miR-125b-1-3p expression level ratio and hsa-miR-6798-5p expression level ratio, respectively, were compared in the expression level.

[0115]

　As a result, the expression level ratio of hsa-miR-6798-5p is 0.95, but exceeds the threshold value 0.8, the expression level ratio of hsa-miR-125b-1-3p is 0.73, below the threshold 0.8. Since one of the two reference miRNA is below the threshold value, the quality of the miRNA to be included in the sample was determined to be defective.

[0116]

　On the other hand, the expression amount of all miRNA detected, the correlation coefficient between the expression amount of all miRNA from standard bodily fluid analyte is somewhat low as 0.94, the decomposition of the miRNA occurs, the quality was somewhat worse It has been shown. This was consistent with the determination result of the quality due to the present invention.

[0117]

　The evaluation method of the quality of the miRNA of the present invention, for comparison with the method according to the electrophoresis is a conventional quality evaluation method, the quality by electrophoresis using serum specimens used in Examples 4 and 5 evaluation of were carried out.

[0118]

　As a result, after a lapse of the stand 72 hours, RNA was extracted from serum samples at stand 168 hours elapsed, RNA degradation of the nucleic acid sample is extracted from commercial serum specimens, which is a standard fluid sample that is not progressed compared with, the result of the electrophoresis could not be confirmed is the difference between the quality (the degree of decomposition).

[0119]

　as a reference miRNA used for the determination of the quality, instead of hsa-miR-125b-1-3p (SEQ ID NO: 2), to change to using the hsa-miR-149-3p (SEQ ID NO: 25) also the serum samples, was changed to the after standing 168 hours at 4 ° C. after serum preparation, other than these in the same manner as in example 2, these two miRNA derived and from the standard bodily fluid analyte serum samples the amount of expression was measured. Here, hsa-miR-149-3p was used as a reference miRNA, in the first embodiment, one is a miRNA whose expression level was most stable without change (decrease) by lapse of standing time .

[0120]

　As a result, the expression level ratio was 0.98, higher than the threshold value 0.8. When evaluated in the same criteria and methods of the present invention, the quality of miRNA contained in the specimen was determined to be good. However, the expression level of all miRNA from the detected serum specimens, since the correlation coefficient between the expression amount of all miRNA from standard bodily fluid analyte was 0.94, actually occurs decomposition of miRNA, quality it has been shown that worsening had. That is, when the reference miRNA that can be used in the present invention using the hsa-miR-149-3p no, can not correctly determine the quality.

[0121]

　In the same manner as in Example 2, as a reference miRNA used for the determination of the quality, in addition to using the hsa-miR-125b-1-3p (SEQ ID NO: 2), does not depend on the degradation of RNA non is an exploded endogenous miRNA hsa-miR-4463 (SEQ ID NO: 26) is also utilized to measure the expression levels of these two miRNA derived and from the standard bodily fluid analyte serum sample. For each serum sample and the standard bodily fluid analyte, after obtaining the expression level ratio returns dividing the expression level of hsa-miR-125b-1-3p in expression level of hsa-miR-4463, the expression level ratio of from serum samples the calculated ratio of return split expression level ratio expression level ratio from the standard bodily fluid analyte was compared to a threshold.

[0122]

　As a result, hsa-miR-125b-1-3p expression level ratio returned divided by the expression level of hsa-miR-4463 the expression level of, in serum from the specimen is 0.97, the derived standard bodily fluid analyte was 0.98 . The ratio of these expression level ratio of 0.99, since it exceeds the threshold value 0.8, and determine the quality of the miRNA to be included in the sample as good.

[0123]

　On the other hand, the expression amount of all miRNA from the detected serum specimens, the correlation coefficient between the expression amount of all miRNA from standard bodily fluid analyte of 0.99, that the quality of the miRNA was not decomposed better shown It has been. This was consistent with the determination result of the quality due to the present invention.

The scope of the claims

[Requested item 1]

　A method for evaluating the quality of the miRNA from bodily fluid analyte,
　selected prepared from the body fluid sample and standard bodily fluid analyte, using an RNA sample comprising miRNA, from miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1-12 is one or more criteria miRNA is to measure the abundance in body fluid sample and standard bodily fluid analyte respectively, the measuring step;
　the abundance measurements or representative values of one or more reference miRNA in a body fluid sample, standard abundance measurements of one or more reference miRNA in bodily fluid analyte or as compared to the representative value, the difference in abundance measurements or representative value of 1 or more criteria miRNA between bodily fluid analyte and standard bodily fluid analyte or obtaining a ratio comparing step; and
　obtained in the comparing step, one or more abundance measurements of the reference miRNA or based on the difference or ratio of the representative value, the quality of the quality of the miRNA from bodily fluid analyte determining, determining step
including the Law.

[Requested item 2]

　Said comparing step, the difference or ratio of the abundance measurements of first reference miRNA, the difference between the representative value of abundance measurements of a plurality of difference or ratio of reference miRNA respective abundance measurements or more reference miRNA, or a step of obtaining a ratio, the method of claim 1, wherein.

[Requested item 3]

　The determination step, the one or more difference or ratio of abundance measurements or the representative value of the reference miRNA, comprising comparing a predetermined threshold as a reference, according to claim 1 or 2 wherein.

[Requested item 4]

　Said comparing step, determining a difference by subtracting the abundance measurements or the representative value in the standard bodily fluid analyte from abundance measurements or the representative value in the body fluid sample, or, abundance measurements in bodily fluid analyte or method according to the any one of claims 1 to 3 comprising determining the abundance measurements or ratio is divided by the representative value of the standard bodily fluid sample the representative value.

[Requested item 5]

　The determining step comprises comparing the difference or ratio of the abundance measurements or the representative value of the one or more reference miRNA, with a predetermined threshold value as a reference, the difference or ratio exceeds a threshold value the method of the quality of miRNA from bodily fluid analyte good and determines, according to claim 4, wherein when.

[Requested item 6]

　Representative value of abundance measurements of a plurality of reference miRNA bodily fluid analyte and during normal body fluid specimen, respectively, an average value or median abundance measurements of a plurality of reference miRNA, any one of claims 1 to 5, the method according to item 1.

[Requested item 7]

　Said measuring step comprises correcting the abundance measurements of one or more criteria miRNA in a body fluid sample and standard bodily fluid analyte, subsequent steps are performed using the corrected measurements, according to claim 1 the method according to any one of 1-6.

[Requested item 8]

　Said measuring step includes a probe for capturing an immobilized the one or more reference miRNA on a support, thereby being extracted from a bodily fluid sample and standard bodily fluid analyte is contacted with a nucleic acid sample which has been labeled with a labeling substance, respectively by performing hybridization Te comprises measuring the abundance of the one or more criteria miRNA bodily fluid analyte and during normal bodily fluid analyte, the method according to any one of claims 1 to 7.

[Requested item 9]

　The measuring step, at the same time as the measurement of the abundance of the one or more reference miRNA in the body fluid sample comprises measuring the abundance of a target miRNA in the body fluid sample, one of claims 1 to 8, 1 the method according to item.

[Requested item 10]

　Said measuring step comprises correcting the abundance measurements of the target miRNA in the body fluid sample, the method of claim 9, wherein.

[Requested item 11]

　Nucleic Acids said measuring step has been immobilized on a support, labeled with the probe and is extracted from the bodily fluid analyte labeling substance for capturing the probe and the one or more reference miRNA for capturing a target miRNA by contacting the sample performing hybridization comprises measuring in a body fluid sample target miRNA and the one or more abundance of reference miRNA, respectively, according to claim 9 or 10 a method according.

[Requested item 12]

　It said body fluid sample is blood, serum or plasma, the method according to any one of claims 1 to 11.

[Requested item 13]

　To assess the quality of the miRNA from bodily fluid analyte, one or a plurality of computers,
　were prepared from the body fluid sample and standard bodily fluid analyte were measured respectively using the RNA samples containing miRNA, in SEQ ID NO: 1-12 It acquires abundance measurements of bodily fluid analyte and in standard bodily fluid analyte of one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence shown, measured value acquisition step;
　one or more criteria miRNA in bodily fluid analyte the abundance measurements or the representative value, as compared with the abundance measurements or representative value of 1 or more criteria miRNA in standard bodily fluid analyte, one or more reference miRNA between bodily fluid analyte and standard bodily fluid analyte obtaining abundance measurements or difference or ratio of the representative values of the comparison; and
　obtained in the comparing step, based on the difference or ratio of abundance measurements or representative values of one or more reference miRNA , from body fluids specimen of miRNA Determining the quality of the quality determination process
program for executing.

[Requested item 14]

　It was recorded according to claim 13 of the program, a computer-readable recording medium.

[Requested item 15]

　Probe for capturing one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by SEQ ID NO: 1 to 12 comprising the immobilized support, miRNA quality evaluation chip.

[Requested item 16]

　Probe for capturing one or more reference miRNA is selected from the miRNA comprising the nucleotide sequence represented by the probe and SEQ ID NO: 1-12 for capturing a target miRNA comprises immobilized support, miRNA expression analysis chip.