Methods for evaluating the quality of serum samples

Abstract

Disclosed is a method in which the deterioration of the quality of a serum specimen is detected with high sensitivity by detecting the amount of white blood cells contained in a serum specimen, in particular a post-sampling serum specimen, even if the white blood cell amount is very small. In a method for evaluating the quality of a serum specimen according to the present invention, in order to evaluate the quality of a serum specimen, one or more miRNAs represented by SEQ ID NOs: 1 and 2 are used as the reference miRNA, and the amount of the reference miRNA contained in a serum specimen and the amount of the reference miRNA contained in a standard serum specimen in which there is no white blood cell mixed therein are compared.

Description

[Technical field]

[0001] The present invention relates to a method for assessing the quality of a serum sample (typically, contamination with white blood cells) by the abundance of a specific miRNA contained in the serum sample. [Background technology]

[0002] miRNA (microRNA) is transcribed from genomic DNA as RNA (precursor) with a hairpin-like structure. This precursor is cleaved by a specific enzyme, dsRNA cleavage enzyme (Drosha, Dicer) with RNase III cleavage activity, and then changes to a double-stranded form, and then becomes single-stranded. One of the antisense strands is incorporated into a protein complex called RISC, and is thought to be involved in the translational inhibition of mRNA. Thus, since the form of miRNA differs at each stage after transcription, it is usually necessary to consider various forms such as hairpin structure, double-stranded structure, and single-stranded structure when detecting miRNA. miRNA consists of RNA of 15 to 25 bases, and its existence has been confirmed in various organisms.

[0003] In recent years, it has been suggested that miRNAs are not only present in cells, but also in bodily fluids such as serum, plasma, urine, and spinal fluid, which are samples that do not contain cells, and that their abundance may serve as a biomarker for various diseases, including cancer. As of July 2019, there are more than 2,600 types of miRNAs in humans, and when a measurement system such as a highly sensitive DNA microarray is used, it is possible to simultaneously detect the expression of more than 1,000 types of miRNAs in serum and plasma. Therefore, biomarker discovery research is being conducted using DNA microarray methods for bodily fluids such as serum, plasma, urine, and spinal fluid, and it is expected that this will be developed into a biomarker test that can detect diseases early.

[0004] On the other hand, blood contains RNA derived from blood cells such as platelets, white blood cells, and red blood cells. When gene expression analysis is performed using DNA microarrays, it is known that miRNA derived from these blood cells affects the measurement of miRNA derived from body fluids such as serum, plasma, and urine. In particular, white blood cells are blood cells with cell nuclei, and if white blood cells are mixed into a sample, it becomes difficult to accurately measure the expression level of the target miRNA due to the influence of nucleic acids contained in the cell nuclei. In tests that measure the amount of miRNA present in body fluids as a biomarker for disease, if tests and diagnoses are made based on the measured amount of the amount with uncertainty, opportunities for appropriate treatment will be missed, or incorrect medical treatment will be applied, imposing unnecessary economic and physical burdens on patients. Therefore, when testing using serum or plasma samples, it is extremely important to use samples in which the miRNA in white blood cells is not included in the target miRNA in order to accurately measure the amount of the target miRNA.

[0005] Conventionally, blood cell analyzers have been used to measure the amount of white blood cells in blood. For example, the XT-2000i (Sysmex Corporation) can measure the number of white blood cells in a blood sample based on their size and cell density. Another method proposed for measuring white blood cells is ELISA, which uses a specific antibody against the white blood cell-specific CD45 surface antigen.

[0006] However, the above-mentioned method using the blood cell analyzer has a measurement limit of about 1.25 million cells per 500 μl, so if a sample contains a smaller amount of white blood cells than this limit, the measurement sensitivity is insufficient and the sample may be erroneously determined to contain no white blood cells. Therefore, this method cannot be an effective method for measuring sample quality. In addition, the evaluation method using ELISA, which uses white blood cell-specific surface antigens as an indicator, cannot be an effective method for measuring sample quality because even fragments that do not contain white blood cell-derived RNA are erroneously measured due to the remaining surface antigens.

[0007] On the other hand, Patent Document 1 describes miRNAs enriched in leukocytes, and describes that three types of miRNAs, miR-342-3p, miR-150-5p, and miR-146a-5p, can serve as leukocyte markers. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special table number 2017-521051 Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, in order to accurately measure the amount of RNA present in a target serum sample, it is important to confirm that the serum sample does not contain white blood cells. For example, it is required to detect the presence of a very small amount of white blood cells, about 1000 cells, in 300 μL of serum sample. For example, when performing gene expression analysis using a DNA microarray, particularly when the deterioration of the sample quality due to the presence of a very small amount of white blood cells affects the measurement and diagnosis of the target gene, a sensitive indicator or method that can detect the presence of a very small amount of blood cells with high sensitivity and therefore determine whether expression analysis can be performed is required.

[0010] As described above, it is possible that the presence of white blood cells may be detected by measuring the expression levels in a sample of the three miRNAs shown in Patent Document 1. However, as shown in the Examples described below, when the expression levels in serum were measured using a DNA microarray, the expression levels were all low, and no change was observed even in samples contaminated with a very small amount of white blood cells, indicating that the quality of serum samples cannot be measured at the desired level even if these miRNAs are used.

[0011] For example, Patent Document 1 states that miR-342-3p can be used as a white blood cell marker. However, when using a sample containing a very small amount of white blood cells (approximately 1,000 cells) in 300 μL of serum, the result may be erroneously given as no white blood cells are present, and therefore using fluctuations in miR-342-3p to measure the quality of a sample is not an effective method.

[0012] An object of the present invention is to find a method for detecting even a very small amount of white blood cells contained in a serum sample, particularly a serum sample after collection, and for sensitively detecting a deterioration in the quality of the serum sample. [Means for solving the problem]

[0013] In order to solve the above problems, the present inventors discovered that a miRNA whose abundance changes upon addition of leukocytes after collection of a sample (hereinafter referred to as a "reference miRNA") can be used as a miRNA that can be detected even when a very small amount of leukocytes is present, and that the quality of a serum sample can be evaluated by measuring the abundance of the miRNA, thus completing the present invention. That is, the present invention is a method for evaluating the quality of a serum sample by using one or more miRNAs among the miRNAs shown in SEQ ID NOs: 1 to 2 as a reference miRNA, and comparing the abundance of the miRNA contained in the serum sample with the abundance of the miRNA contained in a standard serum sample not contaminated by leukocytes, and includes the following aspects.

[0014] (1) Serum samples White blood cell contamination A method for evaluating a measuring step of measuring the abundance of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 in a serum sample and in a standard serum sample; a comparison step of comparing the abundance or index value of the one or more types of reference miRNA contained in the serum sample obtained in the measurement step with the abundance or index value of the one or more types of reference miRNA contained in a standard serum sample to obtain a difference or ratio in the abundance or index value of the one or more types of reference miRNA between the serum sample and the standard serum sample; and Based on the difference or ratio of the abundance or index value of one or more kinds of reference miRNA obtained in the comparison step, Presence or absence of white blood cell contamination A judgment step for judging The method comprising: (2) In the determination step, if the difference or ratio between the abundance or index value of one or more types of reference miRNA contained in the serum sample and the abundance or index value of one or more types of reference miRNA contained in the standard serum sample exceeds a predetermined threshold value as a criterion, the serum sample is White blood cells are mixed in The method according to (1), (3) The method according to (1) or (2), wherein the measuring step comprises contacting a probe for capturing one or more reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 immobilized on a support with a nucleic acid sample derived from a serum specimen and a nucleic acid sample derived from a standard serum specimen, both of which are labeled with a labeling substance, to perform hybridization, and measuring the amount of the one or more reference miRNAs present in the serum specimen and the standard serum specimen. (4) The method according to any one of (1) to (3), further comprising a correction step of correcting the measured values ​​of the abundance of the one or more types of reference miRNA obtained in the measurement step, and the determination step is carried out using the corrected abundance values. (5) The method according to any one of (1) to (4), wherein the measuring step comprises measuring the abundance of one or more types of reference miRNA in a serum sample and simultaneously measuring the abundance of a target miRNA in the serum sample. (6) The method according to (5), wherein the measuring step comprises contacting a probe for capturing a target miRNA immobilized on a support and a probe for capturing one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 with a nucleic acid sample derived from a serum specimen labeled with a labeling substance to perform hybridization, and measuring the abundance of the target miRNA and the one or more types of reference miRNA in the serum specimen, respectively. (7) The method according to (5) or (6), further comprising a correction step of correcting the measured values ​​of the abundance of the target miRNA in the serum sample and the measured values ​​of the abundance of the one or more types of reference miRNA obtained in the measurement step. (8) Serum samples White blood cell contamination In order to evaluate a measurement value acquisition step of acquiring the measurement values ​​of the abundances of one or more types of reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 in the serum sample and the standard serum sample, the measurement values ​​being measured using an RNA sample prepared from a serum sample and a standard RNA sample prepared from a standard serum sample; A comparison step of comparing the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNAs in a serum sample with the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNAs in a standard serum sample to obtain a difference or ratio of the abundance measurement values ​​or index values ​​thereof of one or more reference miRNAs between the serum sample and the standard serum sample; and Based on the difference or ratio between the abundance measurements or index values ​​of one or more types of reference miRNA obtained in the comparison step, Presence or absence of white blood cell contamination A judgment step for judging A program for executing. (9) A computer-readable recording medium having the program described in (8) recorded thereon. (10) A support having immobilized thereon a probe for capturing one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2. Found in serum samples miRNA To assess the miRNA contamination in leukocytes Tips. (11) Serum samples White blood cell contamination An apparatus for evaluating a storage means for storing the measured values ​​of the abundance of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 in the serum specimen and the standard serum specimen, the measured values ​​being obtained using an RNA sample prepared from a serum specimen and a standard RNA sample prepared from a standard serum specimen; a comparison means for comparing the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA in a serum sample with the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA in a standard serum sample to obtain a difference or ratio between the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA between the serum sample and the standard serum sample; Based on the difference or ratio between the abundance measurements or index values ​​of one or more types of reference miRNA obtained by the comparison means, Presence or absence of white blood cell contamination A determination means for determining The apparatus comprising: Effect of the Invention

[0015] According to the present invention, it is possible to evaluate the degree of deterioration in the quality of a serum sample with high accuracy and in a simple manner, and in particular, it is possible to evaluate whether deterioration in sample quality (mainly contamination with blood cells) has occurred due to differences in blood collection conditions, such as the blood collection tube used, after collection of the sample, which was difficult with conventional methods. Furthermore, according to the present invention, it is possible to evaluate with high accuracy and in a simple manner whether a serum sample has a quality suitable for gene expression analysis using, for example, miRNA, and therefore it is possible to obtain more accurate test results in disease tests using the amount of a biomarker present in a serum sample as an index. [Brief description of the drawings]

[0016] [Figure 1] 1 shows the changes in the abundance of two types of miRNA when leukocytes were added to serum (two conditions), as detected by DNA microarray in Example 1. [Diagram 2] 1 shows the change in the sum of the abundances of two types of miRNA when leukocytes are added to serum, as detected by DNA microarray in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention is a method for evaluating the quality of a serum sample, comprising the steps of: a measurement step of measuring the abundance of one or more miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 as reference miRNAs in a serum sample and a standard serum sample; a comparison step of obtaining a difference or ratio between the abundance or index value of one or more types of reference miRNA in the serum sample obtained in the measurement step and the abundance or index value of one or more types of reference miRNA in the standard serum sample; and an assessment step of judging the quality of the serum sample based on the difference or ratio obtained in the comparison step.

[0018] The method of the present invention can be used to determine the suitability of performing gene expression analysis, such as analysis using an array chip such as a microarray, polymerase chain reaction (PCR), or sequence analysis, by evaluating the quality of miRNA contained in a serum sample in advance. Gene expression analysis includes, for example, labeling miRNA in serum, measuring the amount of each miRNA present using a support on which a probe for capturing one or more target miRNAs and a probe for capturing a reference miRNA are immobilized, performing an amplification reaction using a primer for amplifying one or more target miRNAs and a primer for amplifying a reference miRNA, and measuring the amount of target miRNA present. Furthermore, these results can be used to analyze or test gene expression, for example, to test for measuring gene expression in a clinical sample to understand the pathology.

[0019] "miRNA" is a type of non-coding RNA (ncRNA) that means short-stranded RNA with a length of about 15 to 25 bases produced in vivo, and is thought to have the function of regulating the expression of mRNA. miRNA is transcribed from genomic DNA as RNA (precursor) with a hairpin-like structure. This precursor is cleaved by a specific enzyme, dsRNA cleavage enzyme (Drosha, Dicer), which has RNase III cleavage activity, and then changes to a double-stranded form, and then becomes single-stranded. One of the antisense strands is then incorporated into a protein complex called RISC, and is thought to be involved in the translational inhibition of mRNA. Thus, since the form of miRNA differs at each stage after transcription, it is usually necessary to consider various forms such as hairpin structures, double-stranded structures, and single-stranded structures when targeting miRNA (detection subject). The presence of miRNA has been confirmed in various organisms.

[0020] The specimen to which the present invention can be applied is a serum specimen separated and prepared from a living body. The type of living body from which the serum specimen is derived is not particularly limited and includes various biological species, but is typically a mammal, particularly a human.

[0021] Serum samples contain various biomolecules, such as proteins, peptides, nucleic acids such as DNA and RNA, and metabolites. These biomolecules are suitable as biomarkers for various diseases.

[0022] The quality of a serum sample is reduced (deteriorated) or poor, and refers to a change in the amount of the above-mentioned biomolecules from the amount originally present in the whole blood sample at the time of blood collection and reflected in the serum sample after preparation, and mainly refers to an increase in the amount of miRNA in white blood cells mixed into the RNA sample extracted from the serum sample. In determining whether the quality of a serum sample is good or bad using the method of the present invention, "the quality of the serum sample is poor" mainly refers to a state in which the RNA sample extracted from the serum sample is mixed with miRNA in white blood cells, or the amount of the mixed-in amount is so large that it cannot be said to be very small, and "the quality of the serum sample is good" mainly refers to a state in which the RNA sample extracted from the serum sample is not mixed with miRNA in white blood cells, or the amount of the mixed-in amount is suppressed to a very small amount. In the present invention, the term "miRNA contained in a serum sample" means miRNA contained in an RNA sample extracted (prepared) from a serum sample, and when blood cells are mixed into the serum sample, the miRNA contained in the blood cells is also included in "miRNA contained in a serum sample". In the present invention, the term "quality of miRNA contained in a serum sample" can be used interchangeably with "quality of a serum sample." Possible causes of deterioration in the quality of a serum sample include blood collection conditions such as the type of blood collection tube (e.g., the presence or absence of a separating agent), the centrifugation speed, and the type of blood collection needle, as well as temperature, heat, external forces such as vibration and ultrasound on the sample, and various direct and indirect physical forces including electric fields and magnetic fields, but the causes of deterioration in quality are not limited to these.

[0023] In the present invention, RNA can be extracted from these samples, and the amount of miRNA present can be measured using this RNA. For RNA extraction, known methods (e.g., the method of Favaloro et al. (Favaloro et al., Methods Enzymol. 65: 718 (1980)) or various commercially available kits for RNA extraction (e.g., Qiagen's miRNeasy, Toray Industries, Inc.'s "3D-Gene" RNA extraction reagent from liquid sample, etc.) can be applied.

[0024] <Measurement process> In the present invention, the abundance of one or more reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 in a serum sample and a standard serum sample is measured. In addition, the abundance of a target miRNA in the serum sample may be measured simultaneously with the abundance of the reference miRNA in the serum sample. The target miRNA is defined as a miRNA that is to be measured for each purpose among the miRNAs contained in the serum sample.

[0025] The standard serum specimen is a serum specimen in which the quality deterioration defined above has not progressed, and is a serum specimen in which blood cells such as white blood cells are not mixed in or are suppressed to a very small amount. For example, a serum specimen that is collected under the same blood collection conditions as the serum specimen to be evaluated for quality and is frozen and stored immediately after preparing the serum, or a commercially available serum specimen can be used as the standard serum specimen. The serum specimen may be derived from the same individual as the test serum specimen, or may be derived from a different individual of the same species.

[0026] The miRNAs consisting of the base sequences shown in SEQ ID NOs: 1 and 2, which can be used as reference miRNAs in the present invention, are miRNAs that have been discovered by the present inventors as miRNAs whose abundance changes depending on changes in the quality of a serum sample. When the quality of a serum sample changes (deteriorates), the abundance of each gene RNA contained in the sample changes. In this case, for all genes detected by gene expression analysis, the correlation between the RNA in a serum sample that has been deteriorated due to contamination with blood cells or the like (deteriorated serum sample) and the RNA in the freshest serum sample that has not deteriorated (standard serum sample) decreases, for example, with the correlation coefficient being 0.96 or less. The extent to which the quality of a deteriorated serum sample has deteriorated can be determined, for example, by the abundance ratio of each miRNA (FC i) can be used for evaluation. In the present invention, this 2SD value is referred to as the overall variation index value. When the overall variation index value is 0.5 or more, it means that the degree of variation in the abundance of each miRNA measured in the degraded serum sample is large, and therefore the degree of quality deterioration of the degraded serum sample is large. The reference miRNA used in the present invention is a miRNA whose abundance varies in correlation with such overall RNA variation.

[0027]

number

[0028]

number

[0029] Here, in Equation 1 and Equation 2, miRNA i_control is the abundance of the i-th miRNA in a standard serum sample, miRNA i_sample is the abundance of the i-th miRNA in the degraded serum sample, FC 平均値 is the average abundance ratio of n miRNAs (abundance in standard serum samples / abundance in degraded serum samples), It is.

[0030] As the reference miRNA used in the present invention, a miRNA whose abundance changes depending on the type of blood collection tube (presence or absence of a separating agent, etc.) and centrifugation conditions can be selected. The miRNA whose abundance changes depending on the blood collection tube and centrifugation conditions in serum state can be selected, for example, by separating white blood cells and serum from whole blood collected, adding white blood cells to the serum under certain conditions (for example, 1,000 white blood cells, 10,000 white blood cells), intentionally degrading the serum to prepare a degraded serum sample, measuring the abundance of miRNA in the degraded serum sample and a non-degraded serum sample to which no white blood cells have been added, and comparing the degree of change. The abundance of miRNA obtained from the degraded serum sample is compared with the abundance of miRNA obtained from the non-degraded serum sample, and a miRNA with a difference is selected, whereby a reference miRNA can be selected. Generally, in DNA microarray measurement, a two-fold change in abundance is considered to be a sufficient difference, so it is preferable to select a miRNA with a two-fold or greater difference between the degraded serum sample and the non-degraded serum sample.

[0031] In the measurement step of the present invention, the amounts of one or more reference miRNAs selected from the miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 present in a serum sample and a standard serum sample are measured.

[0032] Hereinafter, the probes for capturing the reference miRNA and the target miRNA are collectively referred to as "capture probes" or simply as "probes."

[0033] The amount of miRNA present in serum samples and standard serum samples can be measured by, for example, a hybridization assay using an array chip such as a microarray on which a probe that specifically binds to a target miRNA is immobilized. In the present invention, an array chip containing a support on which a "reference miRNA capture probe" for capturing one or more reference miRNAs is immobilized can be used. Alternatively, an array chip containing a support on which a "target miRNA capture probe" for capturing a target miRNA is further immobilized can be used.

[0034] The term "capture probe" or "probe for capture" refers to a substance that can bind directly or indirectly, preferably directly and selectively, to the miRNA to be captured. Representative examples include nucleic acids, proteins, sugars, and other antigenic compounds. In the present invention, a nucleic acid probe can be preferably used. As the nucleic acid, in addition to DNA and RNA, nucleic acid derivatives such as PNA (peptide nucleic acid) and LNA (locked nucleic acid) can be used. Here, the derivative means, in the case of nucleic acids, chemically modified derivatives such as derivatives labeled with a fluorescent group or the like, derivatives containing modified nucleotides (e.g., nucleotides containing groups such as halogen, alkyl such as methyl, alkoxy such as methoxy, thio, and carboxymethyl, and nucleotides that have undergone base reconstitution, double bond saturation, deamination, and substitution of oxygen molecules with sulfur molecules, etc.).

[0035] From the viewpoint of ensuring the stability and specificity of hybridization, the length of the nucleic acid probe is preferably equal to or longer than the length of the miRNA to be detected. In general, a length of about 17 to 25 bases allows the probe to fully exhibit selective binding to the target miRNA. Such a short oligonucleic acid probe can be easily prepared by known chemical synthesis methods, etc.

[0036] It is known that the stringency during hybridization is a function of temperature, salt concentration, probe chain length, GC content of the nucleotide sequence of the probe, and the concentration of the chaotropic agent in the hybridization buffer. Examples of stringent conditions include those described in Sambrook, J. et al. (1998) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York. Stringent temperature conditions are about 30°C or higher. Other conditions include hybridization time, concentration of detergent (e.g., SDS), and the presence or absence of carrier DNA, and various stringencies can be set by combining these conditions. Those skilled in the art can appropriately determine conditions for obtaining the function of a capture probe prepared for the detection of a desired sample RNA.

[0037] Although the nucleic acid probe is a complementary strand of the miRNA to be captured, it is clear to those skilled in the art that there are sequences other than the target to be captured that bind by cross-hybridization. That is, in the present invention, when the abundance of the reference miRNA is measured using the complementary strand of the reference miRNA shown in SEQ ID NO: 1-2 as a probe, the abundance of the reference miRNA is also detected as a "change in the abundance of the reference miRNA", including the change in the abundance of cross-hybridizing RNA other than the reference miRNA. In the present invention, the quality of the serum sample can be judged by judging the abundance of such cross-hybridizing RNA as well.

[0038] Sequence information of miRNAs can be obtained from databases such as GenBank (http: / / www.ncbi.nlm.nih.gov / genbank / ) and the miRBase website (http: / / www.mirbase.org / ). Reference miRNA capture probes and target miRNA capture probes can be designed based on the sequence information available from these sites.

[0039] The number of miRNA capture probes immobilized on the support is not particularly limited.For example, the number of miRNA capture probes that cover all known miRNAs whose sequences are identified can be immobilized on the support to measure the amount of miRNA present, or the number of desired miRNA capture probes that are immobilized on the support according to the purpose of testing can be used.

[0040] The support on which the capture probes are aligned and immobilized can be the same as the support used in known microarrays, macroarrays, etc., and can be, for example, a slide glass, a membrane, beads, etc. Supports having a shape with multiple convex portions on the surface, as described in Japanese Patent No. 4244788, etc., can also be used. The material of the support is not particularly limited, and examples thereof include inorganic materials such as glass, ceramic, and silicon; and polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and silicone rubber.

[0041] Known methods for immobilizing a capture probe on a support include synthesizing oligo DNA on the surface of the support and dropping pre-synthesized oligo DNA onto the support surface and immobilizing it there.

[0042] Examples of the former method include the method of Ronald et al. (U.S. Pat. No. 5,705,610), the method of Michel et al. (U.S. Pat. No. 6,142,266), and the method of Francesco et al. (U.S. Pat. No. 7,037,659). In these methods, an organic solvent is used during the DNA synthesis reaction, so the support is preferably made of a material that is resistant to the organic solvent. In addition, in the method of Francesco et al., the support is irradiated with light from the back side to control DNA synthesis, so the support is preferably made of a material that is light-transmitting.

[0043] Examples of the latter method include the method by Hirota et al. (Patent No. 3922454) and a method using a spotter. Spotting methods include the pin method, which mechanically contacts the tip of a pin with the solid phase, the inkjet method that uses the principle of an inkjet printer, and the capillary method using a capillary tube. After spotting, post-treatment such as cross-linking by UV irradiation and surface blocking is performed as necessary. In order to fix the oligo-DNA to the surface-treated support surface by covalent bonding, functional groups such as amino groups and SH groups are introduced to the ends of the oligo-DNA. Surface modification of the support is usually performed by treatment with a silane coupling agent having an amino group or the like.

[0044] Hybridization with each miRNA capture probe immobilized on a support is carried out by preparing a nucleic acid sample (nucleic acid sample derived from a specimen) labeled with a labeling substance from RNA extracted from a specimen (specimen RNA) and contacting this labeled nucleic acid sample with a probe. The "nucleic acid sample derived from a specimen" includes not only RNA extracted from a specimen, but also cDNA and cRNA prepared from the RNA by reverse transcription reaction. The labeled nucleic acid sample derived from a specimen may be specimen RNA directly or indirectly labeled with a labeling substance, or may be cDNA or cRNA prepared from specimen RNA directly or indirectly labeled with a labeling substance.

[0045] Methods for binding a labeling substance to a nucleic acid sample derived from a specimen include a method of binding a labeling substance to the 3' end of a nucleic acid sample, a method of binding a labeling substance to the 5' end, and a method of incorporating a nucleotide bound to a labeling substance into a nucleic acid. In the method of binding a labeling substance to the 3' end and the method of binding a labeling substance to the 5' end, an enzyme reaction can be used. For the enzyme reaction, T4 RNA Ligase, Terminal Deoxitidyl Transferase, Poly A polymerase, etc. can be used. For both labeling methods, the method described in "miRNA Experimental Protocols, edited by Shao-Yao Ying, Yodosha, 2008" can be referred to. In addition, various kits for directly or indirectly binding a labeling substance to the end of RNA are commercially available. For example, examples of kits that directly or indirectly bind a labeling substance to the 3' end include the "3D-Gene" miRNA labeling kit (Toray Industries, Inc.), miRCURY miRNA HyPower labeling kit (Exxicon), NCode miRNA Labeling system (Life Technologies, Inc.), and FlashTag Biotin RNA Labeling Kit (Genisphere, Inc.).

[0046] In addition, as in the conventional method, it is also possible to synthesize cDNA or cRNA from sample RNA in the presence of labeled deoxyribonucleotides or labeled ribonucleotides to prepare cDNA or cRNA incorporating a labeled substance, and then hybridize this with a probe on an array.

[0047] In the present invention, the labeling substance that can be used includes various labeling substances that are also used in known microarray analysis. Specific examples include, but are not limited to, fluorescent dyes, phosphorescent dyes, enzymes, and radioisotopes. Preferred are fluorescent dyes that are easy to measure and detect. Specific examples include, but are not limited to, known fluorescent dyes such as cyanine (cyanine 2), aminomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indocarbocyanine (cyanine 5), cyanine 5.5, cyanine 7, and oyster.

[0048] Furthermore, the labeling substance may be a luminescent semiconductor particle, such as cadmium selenium (CdSe), cadmium telluride (CdTe), indium gallium phosphide (InGaP), or silver indium zinc sulfide (AgInZnS).

[0049] The nucleic acid sample derived from the specimen labeled as described above is brought into contact with the miRNA capture probe on the support, and the nucleic acid sample and the probe are hybridized. This hybridization step can be carried out in exactly the same manner as in the past. The reaction temperature and time are appropriately selected according to the chain length of the nucleic acid to be hybridized, but in the case of nucleic acid hybridization, it is usually about 30°C to 70°C for 1 minute to 10 hours. After hybridization and washing, the signal intensity from the labeling substance in each probe immobilization region on the support is detected. The detection of the signal intensity is carried out using a signal reading device appropriate for the type of labeling substance. When a fluorescent dye is used as the labeling substance, a fluorescent microscope, a fluorescent scanner, or the like may be used.

[0050] The measured values ​​of the detected fluorescence intensity are compared with the surrounding noise. Specifically, the measured values ​​obtained from the probe immobilization region are compared with the measured values ​​obtained from other positions, and if the former is higher, it is deemed to have been detected (validated as positive).

[0051] If the detected measured value contains background noise, the background noise may be subtracted. Alternatively, the surrounding noise may be subtracted from the detected measured value as background noise. Alternatively, the method described in "Microarray Data Statistical Analysis Protocol, edited by Wataru Fujibuchi and Katsuhisa Horimoto, Yodosha, 2008" may be used.

[0052] <Correction process> In the present invention, the measured value of the abundance of a reference miRNA obtained in the measurement step may be used as is in the determination step described below. However, for example, when performing gene expression analysis of a target miRNA contained in a serum sample, the measured value may be corrected by various methods exemplified below to obtain a corrected abundance value, which may be used in the determination step.

[0053] As the correction method, a conventional method can be used, for example, a global normalization method, which uses the measured values ​​of all detected miRNAs for correction, and a quantile normalization method. In addition, correction may be performed using housekeeping RNAs such as U1 snoRNA, U2 snoRNA, U3 snoRNA, U4 snoRNA, U5 snoRNA, U6 snoRNA, 5S rRNA, and 5.8S rRNA, or a specific endogenous miRNA for correction, or correction may be performed using an external standard nucleic acid added during RNA extraction or labeling. "Endogenous" means that it is not artificially added to the sample, but is naturally present in the sample. For example, "endogenous miRNA" refers to miRNA that is naturally present in the sample and originates from the organism that provided the sample. When applying the method of the present invention to perform gene expression analysis of target miRNA contained in a serum sample, it is preferable to use a correction method that utilizes an external standard nucleic acid such as a spike control that is not dependent on the sample.

[0054] <Comparison process> The comparison step of the present invention is a step of obtaining the difference or ratio between the abundance or index value of one or more types of reference miRNAs contained in a serum sample and the abundance or index value of one or more types of reference miRNAs contained in a standard body fluid sample, using the abundance amounts in a serum sample and a standard serum sample of one or more types of reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 obtained in the measurement step.

[0055] As described later in the description of the determination step, when one miRNA is used as a reference miRNA among the miRNAs consisting of the base sequences shown in SEQ ID NOs: 1-2, the difference or ratio between the abundance of the reference miRNA contained in the serum sample and the abundance of the reference miRNA contained in the standard serum sample may be obtained and used for the determination. When multiple miRNAs are used as reference miRNAs, the index values ​​of the abundance of the multiple reference miRNAs contained in the serum sample and the index values ​​of the abundance of the multiple reference miRNAs contained in the standard serum sample may be obtained, and the difference or ratio between the abundance of each reference miRNA contained in the serum sample and the abundance of each reference miRNA contained in the standard serum sample may be obtained and used. Alternatively, the difference or ratio between the abundance of each reference miRNA contained in the serum sample and the abundance of each reference miRNA contained in the standard serum sample may be obtained and used. Alternatively, the difference or ratio between the abundance of each reference miRNA contained in the serum sample and the abundance of each reference miRNA contained in the standard serum sample may be added together, and in the next determination step, a determination is made according to a predetermined standard for each reference miRNA, and the quality of the miRNA contained in the serum sample may be determined.

[0056] <Judgment process> The determination step of the present invention is a step of determining the quality of a serum sample using the difference or ratio between the abundance or index value of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 contained in the serum sample obtained in the comparison step and the abundance or index value of one or more types of reference miRNA contained in a standard body fluid sample.

[0057] The quality can be judged by setting a threshold value as a standard for judging the quality of one or more kinds of reference miRNAs contained in a serum sample and a standard serum sample, or the difference or ratio of their index values, in advance, and judging the quality (good or bad) based on whether the threshold value is exceeded. That is, if the amount of the reference miRNA or the difference or ratio of its index value exceeds a predetermined arbitrary threshold, the quality of the serum sample can be judged to be poor, and if the amount of the reference miRNA or the difference or ratio of its index value is equal to or less than the threshold value, the quality of the miRNA contained in the serum sample can be judged to be good. The judgment step in this embodiment can be called a second comparison step in which a comparison with a threshold value is performed.

[0058] In the determination step, the abundance of the reference miRNA or the difference or ratio of its index value obtained in the comparison step may be converted into a logarithm, and the logarithmic value may be used for determination. When performing logarithmic transformation, it is common to convert into a logarithm with a base of 2.

[0059] When one of the miRNAs consisting of the base sequences shown in SEQ ID NOs: 1 to 2 is used as the reference miRNA, in the comparison step, the difference or ratio between the amount of the reference miRNA contained in a serum sample and the amount of the reference miRNA contained in a standard serum sample is determined, and the quality can be determined based on whether this value exceeds a reference threshold value.

[0060] When multiple reference miRNAs among miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 are used as the reference miRNA, the index values ​​of the abundance of the multiple reference miRNAs contained in the serum sample and the index values ​​of the abundance of the multiple reference miRNAs contained in the standard serum sample are obtained, and the quality can be judged based on whether or not the difference or ratio between these index values ​​exceeds a reference threshold. Here, the index value can be the sum, average, or median of the abundance of the multiple reference miRNAs.

[0061] When multiple reference miRNAs among miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 are used as the reference miRNA, the difference or ratio between the abundance in a serum sample and the abundance in a standard serum sample may be calculated for each reference miRNA, and whether or not each reference miRNA exceeds a reference threshold value may be determined. In this case, it is preferable to provide a further judgment criterion by prioritizing or weighting the individual judgments based on the multiple reference miRNAs.

[0062] When one reference miRNA is used, one miRNA may be arbitrarily selected from the miRNAs shown in SEQ ID NO: 1 to 2. It is preferable to select one whose abundance is shown to vary particularly sensitively with the addition of leukocytes in the Examples below, but both hsa-miR-6769b-5p (SEQ ID NO: 1) and hsa-miR-6842-5p (SEQ ID NO: 2) are preferably selected because their abundance varies significantly with the addition of a small amount of leukocytes. In the Examples below, the miRNA whose abundance is shown to vary significantly with the addition of leukocytes can be said to be a miRNA that can particularly sensitively detect deterioration of serum samples that occurs depending on blood collection conditions at clinical testing sites.

[0063] In addition, when performing more rigorous or highly accurate evaluation, it is preferable to use multiple reference miRNAs. When multiple reference miRNAs are used in the present invention, hsa-miR-6769b-5p and hsa-miR-6842-5p may be used in combination.

[0064] Furthermore, for example, when gene expression analysis is the objective and the target miRNA corresponds to any of the miRNAs shown in SEQ ID NOs: 1 to 2, a reference miRNA may be selected from miRNAs excluding the target miRNA.

[0065] The threshold value used as the criterion for judgment can be set arbitrarily depending on the purpose of evaluation, the desired accuracy, etc. For example, the abundance of a reference miRNA contained in a standard serum sample can be set as the threshold value (the following formula 3A).

[0066] When using one reference miRNA, the quality can be determined, for example, by comparing the amount of reference miRNA present in a body fluid sample with the amount of reference miRNA present in a standard serum sample according to the judgment criteria shown in the following formulas 1 to 9.

[0067] As shown in formula 1A, the abundance e of a reference miRNA contained in a serum sample and the abundance E of a reference miRNA contained in a standard serum sample are measured, and the abundance ratio (e / E) is calculated. If this value exceeds a threshold value t1, the quality of the miRNA contained in the body fluid sample can be determined to be poor. The threshold value t1 is preferably 1 or more, for example 1. e / E>t1 (Eq. 1A).

[0068] Furthermore, as shown in Equation 2A, the difference (eE) between the abundance e of a reference miRNA contained in a serum sample and the abundance E of a reference miRNA contained in a standard serum sample is calculated, and if this value exceeds a threshold value t2, the quality of the miRNA contained in the serum sample can be determined to be poor. For example, if the threshold value t2 is set to 0 and the abundance difference (eE) is greater than 0 (positive), the quality of the miRNA contained in the body fluid sample can be determined to be poor. The threshold value t2 is, for example, 0. eE>t2 (Equation 2A).

[0069] Furthermore, the abundance E of the reference miRNA contained in the standard serum sample may be used as the threshold value t3, and in this case, as shown in Equation 3A, the quality of the miRNA contained in the serum sample can be determined to be poor when the abundance e of the reference miRNA contained in the serum sample exceeds the threshold value t3, i.e., the abundance E of the reference miRNA contained in the standard serum sample. This corresponds to the case where the threshold value t2 is set to 0 when Equation 2A is adopted. e>E(=t3) (Equation 3A).

[0070] In the case of considering factors that affect the measurement results in the experimental operation, endogenous miRNA (hereinafter referred to as "stable endogenous miRNA"), which is a miRNA that exists stably and does not depend on the contamination of blood cells, may be used for the determination. Stable endogenous miRNA is a miRNA that is contained in a constant amount in a serum sample regardless of its quality, and preferably, in a serum sample prepared under constant blood collection conditions (constant blood collection tube and centrifugation conditions), a miRNA whose abundance ratio is 0.90 or more, more preferably 0.96 or more, can be selected when comparing before RNA degradation (immediately after the sample is obtained or prepared, when the degradation of the nucleic acid sample contained therein is not progressing) and after RNA degradation (when a certain time has passed since the sample is obtained or prepared, when the degradation of the nucleic acid sample contained therein is expected to progress) in a serum sample prepared under constant blood collection conditions (constant blood collection tube and centrifugation conditions). For example, hsa-miR-4463 consisting of the base sequence shown in SEQ ID NO: 5, etc., can be used as a stable endogenous miRNA. When performing gene expression analysis of target miRNA contained in a serum sample, the "correction endogenous miRNA" used in the correction step can be commonly used as the "stable endogenous miRNA".

[0071] When the quality of miRNA contained in a serum sample is judged to be good or bad using stable endogenous miRNA, for example, as shown in Formula 4A, the abundance ratio (e / c) between the abundance e of a reference miRNA contained in the serum sample and the abundance c of a stable endogenous miRNA, and the abundance ratio (E / C) between the abundance E of a reference miRNA contained in a standard serum sample and the abundance C of a stable endogenous miRNA are calculated, and if the ratio of these two abundance ratios exceeds a threshold value t4, the quality of the miRNA contained in the serum sample can be judged to be good. In this case, the threshold value t4 is preferably 1.

[0072] Alternatively, as shown in Formula 5A, the abundance difference (ec) between the abundance e of a reference miRNA contained in a serum sample and the abundance c of a stable endogenous miRNA, and the abundance difference (EC) between the abundance E of a reference miRNA contained in a standard serum sample and the abundance C of a stable endogenous miRNA are calculated, and if the ratio of these two abundance differences exceeds a threshold value t5, the quality of the miRNA contained in the serum sample can be determined to be poor. In this case, the threshold value t5 is preferably 1. (e / c) / (E / C)>t4 (Eq. 4A) (ec) / (EC)>t5 (Equation 5A).

[0073] As shown in Equation 6A, the abundance ratio (e / c) between the abundance e of a reference miRNA contained in a serum sample and the abundance c of a stable endogenous miRNA, and the abundance ratio (E / C) between the abundance E of a reference miRNA contained in a standard serum sample and the abundance C of a stable endogenous miRNA are calculated, and if the difference between these two abundance ratios exceeds a threshold value t6, the quality of the miRNA contained in the body fluid sample can be determined to be poor. The threshold value t6 is, for example, 0.

[0074] Alternatively, as shown in Equation 7A, the abundance difference (ec) between the abundance e of a reference miRNA contained in a body fluid sample and the abundance c of a stable endogenous miRNA, and the abundance difference (EC) between the abundance E of a reference miRNA contained in a standard serum sample and the abundance C of a stable endogenous miRNA are calculated, and if the difference between these two abundance differences exceeds a threshold value t7, the quality of the miRNA contained in the serum sample can be determined to be poor. The threshold value t7 is, for example, 0. (e / c)-(E / C)>t6 (Equation 6A) (ec)-(EC)>t7 (Equation 7A).

[0075] Alternatively, the abundance ratio (E / C) between the abundance E of the reference miRNA contained in the standard serum sample and the abundance C of the stable endogenous miRNA may be used as the threshold value t8, and in this case, as shown in Formula 8A, when the abundance ratio (e / c) between the abundance e of the reference miRNA contained in the serum sample and the abundance c of the stable endogenous miRNA exceeds the threshold value t8, i.e., the abundance ratio (E / C) between the abundance E of the reference miRNA contained in the standard serum sample and the abundance C of the stable endogenous miRNA, the quality of the miRNA contained in the serum sample can be determined to be poor. This corresponds to the case where the threshold value t6 is set to 0 when Formula 6A is adopted.

[0076] Alternatively, the difference in abundance (EC) between the abundance E of the reference miRNA contained in the standard serum sample and the abundance C of the stable endogenous miRNA may be used as the threshold value t9, and in this case, as shown in Formula 9A, when the difference in abundance (ec) between the abundance e of the reference miRNA contained in the serum sample and the abundance c of the stable endogenous miRNA exceeds the threshold value t9, i.e., the difference in abundance (EC) between the abundance E of the reference miRNA contained in the standard body fluid sample and the abundance C of the stable endogenous miRNA, the quality of the miRNA contained in the serum sample can be determined to be poor. This corresponds to the case where the threshold value t7 is set to 0 when Formula 7A is adopted. e / c>E / C(=t8) (Formula 8A) ec>EC(=t9) (Equation 9A).

[0077] When multiple miRNAs are used as the reference miRNA, the index value of the abundance of multiple reference miRNAs contained in a serum sample and the index value of the abundance of the multiple reference miRNAs contained in a standard serum sample can be obtained, and the difference or ratio between the two index values ​​can be obtained and used. Specifically, in the judgment criteria shown in the above formulas 1A to 9A, the index value r of the abundance of multiple reference miRNAs contained in the serum sample is used instead of the abundance e of the reference miRNA contained in the serum sample, and the index value R of the abundance of multiple reference miRNAs contained in the standard serum sample is used instead of the abundance E of the reference miRNA contained in the standard serum sample, so that the judgment can be made using formulas 1B to 9B. As the index value, the total value, the average value, or the median value of each abundance can be used. r / R>t1 (Equation 1B) rR>t2 (Formula 2B) r>R(=t3) (Formula 3B) (r / c) / (R / C)>t4 (Equation 4B) (rc) / (RC)>t5 (Formula 5B) (r / c)-(R / C)>t6 (Formula 6B) (rc)-(RC)>t7 (Equation 7B) r / c>R / C(=t8) (Formula 8B) rc>RC(=t9) (Equation 9B).

[0078] Here, in the above formulas 1A to 9A and formulas 1B to 9B, taking into consideration experimental error and the like, thresholds t1 to t9 may be given a certain margin of error α, and may be set to "t1±α" to "t9±α", respectively. In this case, the error α may be set arbitrarily, but for example, in formula 2A, α may be set to about 10% of E to give a margin to threshold t2.

[0079] In addition, the abundance value converted into a logarithm may be used as the threshold value of each abundance. In this case, an appropriate threshold may be set according to the conversion. For example, when applying formula 1A, the abundance ratio (e / E) of the reference miRNA may be converted into a logarithmic value, and the threshold t1 may be set according to the conversion. In this case, the difference between the logarithmic values ​​of the abundances e and E is obtained.

[0080] In addition, the difference or ratio between the amount present in a serum sample and the amount present in a standard serum sample for each reference miRNA is calculated, and a judgment is made for each reference miRNA according to the judgment criteria, and the results are combined to judge the quality of the serum sample.

[0081] Specifically, for example, in the judgment for each reference miRNA, if the number of reference miRNAs judged as good exceeds the number of reference miRNAs judged as bad or any predetermined number, the quality of the serum sample can be judged as good. Conversely, if the number of reference miRNAs judged as bad exceeds the number of reference miRNAs judged as good or a predetermined number, the quality of the serum sample can be judged as bad. In addition, when performing a more rigorous or highly accurate evaluation, the quality of the serum sample may be judged as bad if the judgment result of a specific type of reference miRNA is bad.

[0082] Furthermore, for example, when gene expression analysis is the objective and the target miRNA in the analysis corresponds to any of the miRNAs shown in SEQ ID NOs: 1 to 2, a reference miRNA may be selected from miRNAs excluding the target miRNA.

[0083] The present invention also provides a method for evaluating the quality of a serum sample according to the above-described method for evaluating the quality of a serum sample of the present invention, comprising: a measurement value acquisition step of acquiring the measurement values ​​of the abundances of one or more types of reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 in the serum sample and the standard serum sample, the measurement values ​​being measured using an RNA sample prepared from a serum sample and a standard RNA sample prepared from a standard serum sample; A comparison step of comparing the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNAs in a serum sample with the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNAs in a standard serum sample to obtain a difference or ratio between the abundance measurement values ​​or index values ​​thereof of one or more reference miRNAs between the serum sample and the standard serum sample; and a determination step for determining whether the quality of the serum sample is good or bad based on the difference or ratio between the abundance measurement values ​​of one or more types of reference miRNA or their index values ​​obtained in the comparison step; The present invention provides a program for carrying out the above (i.e., including instructions for causing one or more computers to carry out each of the above steps), and a computer-readable recording medium having the program recorded thereon.

[0084] For example, the program may be incorporated into a miRNA expression analysis device that analyzes the expression level of a desired target miRNA, and in the measurement acquisition step, measurements of the abundance of a reference miRNA in a serum sample and a standard serum sample measured by an expression measurement unit included in the device or an expression measurement device separate from the device may be acquired, and each step may be performed using the measurements. In the measurement acquisition step, in addition to the measurements of the abundance of the reference miRNA, measurements of the abundance of one or more target miRNAs to be subjected to expression analysis in a serum sample measured by the expression measurement unit or expression measurement device may also be acquired. The acquisition of the measurements of one or more target miRNAs may be performed simultaneously with the acquisition of the measurements of the reference miRNA, or may be performed according to the quality judgment result by the judgment means (i.e., when the judgment result is good quality). Each measurement value acquired may be a corrected measurement value. Alternatively, the program may include an instruction to cause a computer to execute a process of correcting the acquired measurement value. Details of each step are as described above with respect to the method for evaluating the quality of a serum sample of the present invention. When the quality is judged to be good, the miRNA expression level analyzer may be configured to execute the expression analysis of one or more target miRNAs and output the analysis results to a monitor, etc. Alternatively, the expression analysis of the target miRNA may be executed simultaneously with or sequentially to the quality evaluation of the serum sample, and when the quality is judged to be good, the expression analysis results may be output with a message indicating that the expression analysis results of the target miRNA are reliable, and when the quality is judged to be poor, the expression analysis results may be output with a message indicating that the expression analysis results of the target miRNA are unreliable or low reliable.

[0085] The present invention also provides an apparatus for evaluating the quality of a serum sample according to the above-mentioned method for evaluating the quality of a serum sample of the present invention (hereinafter, referred to as a quality evaluation apparatus). a storage means for storing the measured values ​​of the abundance of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 and 2 in the serum specimen and the standard serum specimen, the measured values ​​being obtained using an RNA sample prepared from a serum specimen and a standard RNA sample prepared from a standard serum specimen; a comparison means for comparing the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA in a serum sample with the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA in a standard serum sample to obtain a difference or ratio between the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA between the serum sample and the standard serum sample; a determination means for determining whether the quality of a serum sample is good or bad based on the difference or ratio between the abundance measurement values ​​of one or more types of reference miRNA or their index values ​​obtained by the comparison means; Includes.

[0086] The measured values ​​of the abundance of one or more kinds of reference miRNAs in a serum sample and a standard serum sample stored in the storage means are measured by an expression measurement unit included in the quality evaluation device or an expression measurement device separate from the quality evaluation device. In addition to the measured values ​​of the abundance of one or more kinds of reference miRNAs, the measured values ​​of the abundance of one or more target miRNAs to be subjected to expression analysis in a serum sample measured by the expression measurement unit or expression measurement device may be stored. Details of the measurement of miRNAs in a sample are as explained in the <Measurement step> of the method for evaluating the quality of a serum sample of the present invention.

[0087] Each measurement value stored in the storage means may be a corrected measurement value. Alternatively, the quality evaluation device may further include a correction means for executing a process for correcting the measurement value. Details of the correction are as described in the <Correction Step> of the method for evaluating the quality of a serum sample of the present invention.

[0088] The comparison means is a means for carrying out the comparison step in the method for evaluating the quality of a serum sample of the present invention. Details are as described in the <Comparison step>.

[0089] The determining means is a means for carrying out the determining step in the method for evaluating the quality of a serum sample of the present invention, the details of which are as described in the <determining step>.

[0090] The determination means can be referred to as a quality output means that determines whether the quality of a serum sample is good or bad based on the difference or ratio between the abundance measurement values ​​of one or more types of reference miRNA or their index values ​​obtained by the comparison means, and outputs the determined quality. The quality is typically output to a display unit such as a monitor of the device, but it can also be configured to output the comparative analysis results and statistical analysis results to an external storage device such as a database located outside the device via a network.

[0091] The quality may be judged by a means for determining whether the quality is good or bad based on whether the difference or ratio of the abundance or index value of one or more kinds of reference miRNA contained in a serum sample and a standard serum sample exceeds a preset threshold value. The judgment means in this embodiment may be called a second comparison means for comparing with the threshold value.

[0092] The quality evaluation device may be incorporated as a quality evaluation unit in a miRNA expression analysis device that analyzes the expression level of a desired target miRNA, and may be a part of the miRNA expression analysis device. In this case, the measurement value stored in the storage means is a measurement value measured by an expression measurement unit included in the miRNA expression analysis device or an expression measurement device separate from the device. As described above, the measurement value stored in the storage means may be a corrected measurement value, or the miRNA expression analysis device may further include a correction means.

[0093] The miRNA expression level analyzer may be configured to execute the expression analysis of one or more target miRNAs and output the analysis results when the quality is determined to be good by the determination means. Alternatively, the expression analysis of the target miRNA may be executed simultaneously with or sequentially to the quality evaluation of the serum sample, and when the quality is determined to be good, the expression analysis results are output with a clear indication that the expression analysis results of the target miRNA are reliable, and when the quality is determined to be poor, the expression analysis results are output with a clear indication that the expression analysis results of the target miRNA are unreliable or low reliable.

[0094] A "program" is a data processing method written in any language or description method, and may be in any format, such as source code or binary code. Note that a "program" is not necessarily limited to a single configuration, but also includes a distributed configuration consisting of multiple modules or libraries, or a program that works with a separate program, such as an OS (Operating System), to achieve its function. The specific configuration for reading the recording medium, the reading procedure, or the installation procedure after reading may be any well-known configuration or procedure.

[0095] The "recording medium" may be any "portable physical medium" (non-transient recording medium) such as a flexible disk, a magneto-optical disk, a ROM, an EPROM, an EEPROM, a CD-ROM, an MO, a DVD, etc. Alternatively, it may be a "communication medium" that retains a program for a short period of time, such as a communication line or carrier wave when transmitting a program via a network, such as a LAN, a WAN, or the Internet.

[0096] The present invention also provides a chip for evaluating miRNA quality, comprising a support on which probes for capturing one or more types of reference miRNA selected from miRNAs having the nucleotide sequences shown in SEQ ID NOs: 1 to 2 are immobilized. The chip may further comprise one or more probes for capturing target miRNA, so that the quality of miRNA (of a serum sample) can be evaluated during miRNA expression analysis of a serum sample. Here, the target miRNA, one or more types of reference miRNA selected from miRNAs having the nucleotide sequences shown in SEQ ID NOs: 1 to 2, the probes for capturing them, and the support on which these capture probes are immobilized are as described above.

[0097] The chip of the present invention may further have a correction nucleic acid, such as a housekeeping RNA used in the correction step, a specific endogenous miRNA for correction, or an external standard nucleic acid to be added, particularly a probe for capturing the endogenous miRNA for correction, immobilized on a support.

[0098] Hereinafter, publicly known information and the like regarding miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, which can be used as reference miRNAs in the present invention, will be described.

[0099] The term "miR-6769b-5p gene" or "miR-6769b-5p" used as a reference miRNA includes the hsa-miR-6769b-5p gene (miRbase Accession No. MIMAT0027620) consisting of the nucleotide sequence shown in SEQ ID NO: 1, and other biological species homologs or orthologues. The hsa-miR-6769b-5p gene can be obtained by the method described in Ladwig E et al., 2012, Genome Res., vol. 22, pp. 1634-1645. In addition, the "hsa-miR-6769b-5p" is known to have a hairpin-like structure as its precursor, the "hsa-mir-6769b gene" (miRbase Accession No. MI0022706, SEQ ID NO: 3). The terms "miR-6769b-5p" and "hsa-miR-6769b-5p" also encompass precursors of such hairpin-like structures.

[0100] The term "miR-6842-5p gene" or "miR-6842-5p" used as a reference miRNA includes the hsa-miR-6842-5p gene (miRbase Accession No. MI0027586) consisting of the base sequence shown in SEQ ID NO: 2, and other biological species homologs or orthologues. It can be obtained by the method described in Ladwig E et al., 2012, Genome Res., vol. 22, p. 1634-1645. In addition, "hsa-miR-6842-5p" is known to have a hairpin-like structure as its precursor, the "hsa-mir-6842 gene" (miRbase Accession No. MI0022688, SEQ ID NO: 4). The terms "miR-6842-5p" and "hsa-miR-6842-5p" also include precursors of such hairpin-like structures. EXAMPLES

[0101] The process of selecting the reference miRNA that varies depending on the quality of the serum sample of the present invention (contamination of white blood cells in the serum sample) will be described in more detail below. However, the present invention is not limited to the following examples. In the following examples, the value obtained by correcting the measured value of the amount of miRNA present in the sample by quantile normalization or the like is expressed as the "expression level" of the miRNA.

[0102] Example 1: Selection of reference miRNAs capable of detecting leukocyte contamination (DNA microarray) The following experiment was carried out using the “3D-Gene” human miRNA oligo chip (compatible with miRBase release 21) manufactured by Toray Industries, Inc.

[0103] (Serum sample preparation) Three healthy subjects each collected blood using one blood collection tube for serum separation and one blood collection tube for plasma separation. After blood collection, the blood collection tube for plasma separation was centrifuged at 800G for 10 minutes at room temperature to obtain white blood cells. The blood collection tube for serum separation was left to stand for 0.5 hours at room temperature (23°C) and then centrifuged at 2300G for 10 minutes at room temperature to obtain serum. 1,000 and 10,000 white blood cells were added to 300 μL of this serum to prepare white blood cell-containing serum, which was then stored at -80°C together with serum without added blood cells (standard conditions).

[0104] (Preparation of sample RNA and measurement of miRNA abundance) The sera prepared as described above and stored in a freezer were thawed at the same time, and the RNA contained in the serum samples (hereinafter referred to as sample RNA) was extracted using the "3D-Gene" RNA extraction reagent from liquid sample kit (Toray Industries, Inc.).

[0105] The obtained sample RNA was labeled using the "3D-Gene" miRNA labeling kit (Toray). The labeled sample RNA was hybridized using the "3D-Gene" miRNA chip (Toray) according to the standard protocol. The DNA microarray after hybridization was subjected to a microarray scanner (Toray) to measure the fluorescence intensity. The scanner was set to 100% laser output and the photomultiplier voltage was set to AUTO.

[0106] The measured values ​​of each miRNA detected by DNA microarray were transformed into logarithms with a base of 2 and corrected by quantile normalization to obtain the expression level of each miRNA.

[0107] (Selection of reference miRNA) The amount of miRNA present in each serum sample obtained as described above was compared, and reference miRNAs were selected by extracting miRNAs whose amount varied greatly depending on the number of added leukocytes.

[0108] First, the expression level of each miRNA in serum without added leukocytes was used as a standard, and the ratio of the expression level of each miRNA in serum to which 1,000 or 10,000 leukocytes were added to 300 μL of serum (expression level when 1,000 or 10,000 cells were added / expression level without addition) was obtained. Next, of the detected miRNAs, the selection was narrowed down to miRNAs that were stably detected in the high expression region, and the standard miRNAs shown in Table 1 were obtained. All three miRNAs described in Patent Document 1 (miR-343-3p, miR-150-5p, miR-146a-5p) were not selected due to low expression.

[0109] Table 1 shows two types of reference miRNAs (SEQ ID NO: 1-2) and the average inter-individual variation in abundance from the reference condition for each condition, as well as the overall variation index value of miRNA in each sample calculated by the above-mentioned formulas 1 and 2. The expression levels of the miRNAs (SEQ ID NO: 1-2) shown in Table 1 increased depending on the amount of white blood cell contamination, and the abundances varied by more than two-fold (more than 1 in terms of the difference in logarithmic value to base 2).

[0110] Generally, in DNA microarray measurements, a two-fold change in abundance is considered to be a sufficient difference. The overall variation index value showed a higher value of 1 or more under conditions with greater addition of white blood cells, confirming a high degree of deterioration in sample quality. From this, it was confirmed that the miRNA in question can be used as a miRNA indicator whose abundance varies depending on the quality of the serum sample. In other words, it was found that the quality of serum samples can be known by measuring the abundance of the reference miRNAs shown in Table 1.

[0111] [Table 1]

[0112] FIG. 1 shows the abundance of two types of reference miRNAs shown in Table 1 under standard conditions and under conditions (2 conditions) in which the amount of white blood cells mixed in was changed. The abundance of both hsa-miR-6769b-5p (SEQ ID NO: 1) and hsa-miR-6842-5p (SEQ ID NO: 2) was sensitively increased by the addition of white blood cells. For example, when using hsa-miR-6769b-5p as the reference miRNA and determining the deterioration of serum sample quality due to the contamination of a serum sample with 1000 or more white blood cells, the abundance of hsa-miR-6769b-5p of 5.0 is set as the threshold, and when the abundance of hsa-miR-6769b-5p in a certain serum sample exceeds that value, it can be determined that the sample is deteriorated, that is, that the sample is of poor quality.

[0113] From the results of this Example 1, specific examples of threshold value t3 when evaluating the quality of a serum sample according to the method of the present invention using a serum sample under standard conditions as a standard serum sample and formula 3A above are shown in Table 2 below. In the example of Table 2, threshold value t3 is set to E±α (α=0.1 to 0.3). When the abundance e of a reference miRNA contained in a serum sample exceeds the corresponding threshold, the serum sample can be determined to be of poor quality.

[0114] [Table 2]

[0115] <Example 2> Detection of deterioration when white blood cells are mixed in using multiple miRNAs It is also possible to determine deterioration in serum sample quality by combining any two types of reference miRNAs rather than a single miRNA.

[0116] The abundances of hsa-miR-6769b-5p (SEQ ID NO: 1) and hsa-miR-6842-5p (SEQ ID NO: 2) under the standard conditions of Example 1 and under conditions containing 1000 or more white blood cells were used. The average abundances of these two types of miRNA were used as the index value of the serum sample under each condition. For example, the index value of serum under the standard conditions is (4.9 + 5.2) / 2 = 5.05.

[0117] The abundance of each of these miRNAs under each condition is as shown in Figure 1, and the index value (i.e., the average value) of the abundance of these two types of miRNAs under the condition of contamination with 1000 or more white blood cells is as shown in Figure 2, resulting in a more sensitive and high value. For example, when determining the deterioration of the quality of a serum sample due to contamination with 1000 white blood cells, the average value of the abundance of hsa-miR-6869b-5p and hsa-miR-6842-5p in a standard serum or the average value ± α is set as a threshold, and when the index value (average value) of the abundance of hsa-miR-6869b-5p and hsa-miR-6842-5p in a certain serum sample exceeds the threshold value, it is possible to determine that the sample is deteriorated, that is, that the sample is of poor quality. This determination mode corresponds to the above-mentioned formula 3B in which the threshold value t3 is set to R or R ± α.

[0118] <Example 3> Blood was collected from one healthy individual and serum samples were prepared. The obtained serum was dispensed in 300 μL aliquots and immediately stored in a freezer set at -80°C. RNA was extracted using the "3D-Gene" RNA extraction reagent from liquid sample kit (Toray). The obtained sample RNA was labeled using the "3D-Gene" miRNA labeling kit (Toray Industries, Inc.), and quantile normalization was performed during labeling to correct the measured amount of miRNA to the expression amount. The labeled sample-derived RNA was hybridized using the "3D-Gene" miRNA chip (Toray Industries, Inc.) according to the standard protocol. The DNA microarray after hybridization was subjected to a microarray scanner (Toray Industries, Inc.) to measure the fluorescence intensity. The scanner was set to 100% laser output and the photomultiplier voltage was set to AUTO. The detected miRNA signal value was corrected by the signal value of the external standard nucleic acid to obtain the expression amount.

[0119] As the standard miRNA used to judge the quality, hsa-miR-6769b-5p (SEQ ID NO: 1) was selected. In addition, serum not contaminated with white blood cells was used as a standard serum sample, and the expression level of hsa-miR-6769b-5p contained in the standard serum sample was obtained in the same manner as described above. The expression level of the miRNA derived from the serum sample was divided by the expression level of the corresponding miRNA derived from the standard serum sample to obtain the expression level ratio of the two. The threshold was set to 1. Since the expression level ratio was 0.93, which was below the threshold, the quality of the miRNA contained in this serum sample was judged to be good.

[0120] On the other hand, the correlation coefficient between the expression level of all miRNAs derived from the detected serum samples and that of all miRNAs derived from the standard body fluid samples was 0.98, a high value exceeding 0.96, indicating that there was no contamination with miRNAs derived from leukocytes. This was consistent with the quality assessment results according to the present invention.

[0121] <Example 4> The serum sample was changed to one in which 1,000 white blood cells were artificially mixed into the serum sample after preparation, and the rest of the procedure was the same as in Example 3 to measure the expression levels of two types of miRNA (hsa-miR-6769b-5p (sequence number 1) and hsa-miR-6842-5p (sequence number 2)) derived from the serum sample and the standard serum sample. The expression levels of both samples were compared using the average expression level of the two types of miRNA as the index value. As a result, the expression level ratio was 2.89, which exceeded the threshold value, and therefore the quality of the miRNA contained in this serum sample was determined to be poor.

[0122] On the other hand, the correlation coefficient between the expression level of all miRNAs derived from the detected serum samples and that of all miRNAs derived from the standard body fluid samples was 0.95, a low value below 0.96, indicating that the quality had deteriorated due to contamination with miRNAs derived from leukocytes. This was consistent with the quality assessment results according to the present invention.

[0123] <Comparative Example 1> In order to compare the method for evaluating the quality of serum samples of the present invention with a conventional quality evaluation method using a blood cell analyzer, a quality evaluation was carried out using a blood cell analyzer (XT-200i (Sysmex Corporation)) for the serum samples used in Example 4. As a result, even when RNA extracted from the serum sample contaminated with 1000 white blood cells was compared with RNA extracted from a standard serum sample not contaminated with white blood cells, the presence or absence of contamination with 1000 white blood cells could not be confirmed from the results of the blood cell analyzer.

[0124] <Example 5> In the same manner as in Example 3, in addition to using hsa-miR-6842-5p (SEQ ID NO: 2) as a reference miRNA for quality judgment, the expression levels of these two miRNAs derived from serum samples and standard body fluid samples were measured using stable endogenous miRNA hsa-miR-4463 (SEQ ID NO: 5). Quality judgment was performed according to the above-mentioned formula 4A. The threshold value was set to 1.

[0125] As a result, the expression ratio, calculated by dividing the expression level of hsa-miR-6842-5p by the expression level of hsa-miR-4463, was 0.035 for serum samples and 0.061 for standard body fluid samples. The ratio of these expression ratios was 0.573, which was below the threshold, and therefore the quality of the miRNA contained in the samples was determined to be good.

[0126] On the other hand, the correlation coefficient between the expression level of all miRNAs derived from the detected serum samples and that of all miRNAs derived from the standard body fluid samples was 0.98, a high value exceeding 0.96, indicating that the quality of the miRNAs contained in the serum samples was good and that they were not contaminated with miRNAs in leukocytes. This was consistent with the quality assessment results according to the present invention.

Claims

1. 1. A method for assessing leukocyte contamination in a serum sample, comprising: a measuring step of measuring the abundance of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 in a serum sample and in a standard serum sample; a comparison step of comparing the abundance or index value of the one or more types of reference miRNA contained in the serum sample obtained in the measurement step with the abundance or index value of the one or more types of reference miRNA contained in a standard serum sample to obtain a difference or ratio in the abundance or index value of the one or more types of reference miRNA between the serum sample and the standard serum sample; and a determination step for determining whether or not the serum sample is contaminated with leukocytes based on the difference or ratio of the abundance of one or more kinds of reference miRNA or their index values ​​obtained in the comparison step; The method comprising:

2. The method according to claim 1, wherein in the determination step, the serum sample is determined to be contaminated with white blood cells if the difference or ratio between the abundance or index value of one or more types of reference miRNA contained in the serum sample and the abundance or index value of one or more types of reference miRNA contained in the standard serum sample exceeds a predetermined threshold value as a criterion.

3. The method according to claim 1 or 2, wherein the measuring step comprises contacting a probe for capturing one or more reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 immobilized on a support with a serum specimen-derived nucleic acid sample and a standard serum specimen-derived nucleic acid sample, both of which are labeled with a labeling substance, to perform hybridization, and measuring the abundance of the one or more reference miRNAs in the serum specimen and the standard serum specimen.

4. The method according to any one of claims 1 to 3, further comprising a correction step of correcting the measured values ​​of the abundance of the one or more types of reference miRNA obtained in the measurement step, and the determination step is carried out using the corrected abundance values.

5. The method according to any one of claims 1 to 4, wherein the measuring step comprises measuring the abundance of one or more types of reference miRNA in a serum sample and simultaneously measuring the abundance of a target miRNA in the serum sample.

6. The method according to claim 5, wherein the measuring step comprises contacting a probe for capturing a target miRNA immobilized on a support and a probe for capturing one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 with a nucleic acid sample derived from a serum specimen labeled with a labeling substance to perform hybridization, and measuring the abundance of the target miRNA and the one or more types of reference miRNA in the serum specimen.

7. The method according to claim 5 or 6, further comprising a correction step of correcting the measured values ​​of the abundance of the target miRNA and the measured values ​​of the abundance of the one or more types of reference miRNA in the serum sample obtained in the measurement step.

8. a step of: a measurement value acquisition step of acquiring the measurement values ​​of the abundance of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 in the serum sample and the standard serum sample, measured using an RNA sample prepared from a serum sample and a standard RNA sample prepared from a standard serum sample; A comparison step of comparing the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNAs in a serum sample with the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNAs in a standard serum sample to obtain a difference or ratio between the abundance measurement values ​​or index values ​​thereof of one or more reference miRNAs between the serum sample and the standard serum sample; and a determination step for determining whether or not the serum sample is contaminated with leukocytes based on the difference or ratio between the abundance measurement values ​​of one or more types of reference miRNA or their index values ​​obtained in the comparison step; A program to implement the above.

9. A computer-readable recording medium on which the program according to claim 8 is recorded.

10. A chip for evaluating the contamination of miRNA in leukocytes with miRNA contained in a serum sample, comprising a support on which a probe for capturing one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 is immobilized.

11. An apparatus for evaluating the presence of leukocytes in a serum sample, comprising: a storage means for storing the abundance measurement values ​​of one or more types of reference miRNA selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 in the serum specimen and the standard serum specimen, the abundance measurement values ​​being measured using an RNA sample prepared from a serum specimen and a standard RNA sample prepared from a standard serum specimen; a comparison means for comparing the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA in a serum sample with the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA in a standard serum sample to obtain a difference or ratio between the abundance measurement values ​​or index values ​​thereof of one or more types of reference miRNA between the serum sample and the standard serum sample; a determination means for determining whether or not the serum sample is contaminated with leukocytes based on the difference or ratio between the abundance measurement values ​​of one or more types of reference miRNA or their index values ​​obtained by the comparison means; The apparatus comprising:

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