JP7627438B2 - Methods for evaluating the quality of serum samples - Google Patents

Description

The present invention relates to a method for assessing the quality of a serum sample (typically, the presence of white blood cell contamination) by the abundance of specific miRNAs contained in the serum sample.

miRNA (microRNA) is transcribed from genomic DNA as a hairpin-like RNA (precursor). 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, when detecting miRNA, it is usually necessary to consider various forms such as hairpin structures, double-stranded structures, and single-stranded structures. miRNA consists of RNA of 15 to 25 bases, and its existence has been confirmed in various organisms.

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 using a measurement system such as a highly sensitive DNA microarray, 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.

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, and 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.

Traditionally, 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 from their size and cell density. Another method that has been proposed is to quantitatively evaluate white blood cells using ELISA, using a specific antibody against the white blood cell-specific CD45 surface antigen.

However, the method using the above blood cell analyzer has a measurement limit of about 1.25 million cells per 500 μl, so if the sample contains a smaller amount of white blood cells than this limit, the measurement sensitivity is insufficient and the sample may erroneously determine that no white blood cells are present. Therefore, this method cannot be an effective method for measuring sample quality. Furthermore, the ELISA evaluation method using white blood cell-specific surface antigens as an indicator cannot be an effective method for measuring sample quality because it erroneously measures the remaining surface antigens even in fragments that do not contain white blood cell-derived RNA.

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

Special Publication No. 2017-521051

As mentioned 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 necessary to detect the presence of a very small amount of white blood cells, about 1,000 cells, in a 300 μL serum sample. For example, when performing gene expression analysis using a DNA microarray, particularly in cases where the deterioration of sample quality due to the presence of a very small amount of white blood cells affects the measurement and diagnostic results of the target gene, a sensitive indicator or method is required that can detect the presence of very small amounts of blood cells with high sensitivity and, as a result, determine whether expression analysis can be performed.

As described above, it is thought that it may be possible to detect the presence of white blood cells by measuring the expression levels of the three miRNAs shown in Patent Document 1 in a sample. 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.

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 indicated as no white blood cells being present, and therefore using fluctuations in miR-342-3p to measure the quality of a sample is not an effective method.

The objective of the present invention is to find a method for sensitively detecting a deterioration in the quality of a serum sample by detecting even minute amounts of white blood cells contained in the serum sample after collection.

In order to solve the above problems, the present inventors discovered that a miRNA whose abundance changes when white blood cells are added 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 white blood cells are 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 white blood cells, and includes the following aspects.

(1) A method for evaluating the presence of leukocytes 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 and 2 in a serum sample and in a standard serum sample;
the method comprising: a comparing step of comparing the abundance or index values thereof of the one or more types of reference miRNA contained in the serum sample obtained in the measuring step with the abundance or index values thereof of one or more types of reference miRNA contained in a standard serum sample to obtain a difference or ratio between the abundance or index values thereof of the one or more types of reference miRNA between the serum sample and the standard serum sample; and a determining step of determining whether or not the serum sample is contaminated by white blood cells based on the difference or ratio between the abundance or index values of the one or more types of reference miRNA obtained in the comparing step.
(2) The method according to (1), wherein 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, it is determined that the serum sample is contaminated with white blood cells .
(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) for evaluating the presence of leukocytes in a serum sample, the one or more computers are
a measurement value acquisition step of acquiring 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 and 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 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 types of reference miRNAs between the serum sample and the standard serum sample; and a determination step of determining whether or not the serum sample is contaminated by white blood cells based on the difference or ratio between the abundance measurement values or index values thereof of one or more types of reference miRNAs obtained in the comparison step.
(9) A computer-readable recording medium having the program described in (8) recorded thereon.
(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;
and a determination means for determining whether or not the serum sample is contaminated with white blood cells based on the difference or ratio of the abundance measurement values or index values thereof of one or more types of reference miRNA obtained by the comparison means.

The present invention makes it possible to evaluate the degree of deterioration in the quality of serum samples with high accuracy and ease, and in particular makes it possible to evaluate whether deterioration in sample quality (mainly contamination with blood cells) has occurred after sample collection due to differences in blood collection conditions, such as the blood collection tube used, which was difficult with conventional methods. In addition, the present invention makes it possible to evaluate with high accuracy and ease whether a serum sample has a quality suitable for gene expression analysis using, for example, miRNA, and therefore makes it possible to obtain more accurate test results in disease tests using the amount of biomarkers present in serum samples as an index.

1 shows the change in the abundance of two types of miRNA when leukocytes were added to serum (two conditions), as detected by DNA microarray in Example 1. 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.

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 a determination step of determining whether the quality of the serum sample is good or bad based on the difference or ratio obtained in the comparison step.

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 sequencing, by evaluating the quality of miRNA contained in a serum sample in advance. Gene expression analysis includes, for example, labeling miRNA in serum and 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, the results of these analyses can be used to analyze or test gene expression, for example, to perform a test to measure gene expression in a clinical sample to understand the pathology.

"miRNA" is a type of non-coding RNA (ncRNA), which means short-stranded RNA with a length of about 15 to 25 bases that is produced in vivo, and is thought to have the function of regulating mRNA expression. miRNA is transcribed from genomic DNA as a hairpin-like RNA (precursor). 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, when targeting miRNA (detection subject), it is usually necessary to consider various forms such as hairpin structures, double-stranded structures, and single-stranded structures. The existence of miRNA has been confirmed in various organisms.

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.

Serum samples contain a variety of biomolecules, including proteins, peptides, nucleic acids such as DNA and RNA, and metabolic products. These biomolecules are suitable as biomarkers for various diseases.

The deterioration or poor quality of a serum sample means that the amount of the above-mentioned biomolecules present in the whole blood sample at the time of blood collection has changed from the amount that should have been reflected in the serum sample after preparation, and mainly means that the amount of miRNA in white blood cells mixed into the RNA sample extracted from the serum sample has increased. In determining whether the quality of a serum sample is good or bad using the method of the present invention, the term "poor quality of the serum sample" 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 sample is so large that it cannot be said to be very small, and the term "good quality of the serum sample" 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 sample is suppressed to a very small amount. In the present invention, the term "miRNA contained in a serum sample" means the 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 the "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.

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.

<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 and 2 in a serum sample and a standard serum sample is measured. 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.

A standard serum sample is a serum sample that has not deteriorated in quality as defined above, and is primarily a serum sample that is free of or contains only trace amounts of blood cells such as white blood cells. For example, a serum sample that has been drawn under the same blood drawing conditions as the subject serum sample whose quality is to be evaluated and that has been frozen and stored immediately after preparing the serum, or a commercially available serum sample can be used as a standard serum sample. The serum sample may be derived from the same individual as the subject serum sample, or may be derived from a different individual of the same species.

The miRNAs consisting of the base sequences shown in SEQ ID NOs: 1 and 2, which can be used as the reference miRNA in the present invention, are miRNAs that have been found 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 (deteriorated serum sample) that has been deteriorated due to contamination with blood cells, etc., and the RNA in the freshest serum sample (standard serum sample) that has not deteriorated decreases, for example, the correlation coefficient becomes 0.96 or less. The degree of deterioration of the quality of a deteriorated serum sample can be evaluated, for example, by using the value of twice the standard deviation (2SD) of the abundance ratio (FC _i ) of each miRNA, which can be calculated by the following formulas 1 and 2. In the present invention, this value of 2SD is called 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 deteriorated serum sample is large, and therefore the degree of deterioration of the quality of the deteriorated serum sample is large. The reference miRNAs used in the present invention are those miRNAs whose abundance changes correlate with such global RNA variations.

Here, in formula 1 and formula 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;
_{The average FC value} is the average value of the abundance ratio of n miRNAs (abundance in standard serum samples/abundance in degraded serum samples).
It is.

As the reference miRNA used in the present invention, it is possible to select miRNAs whose abundance changes depending on the type of blood collection tube (presence or absence of a separating agent, etc.) and centrifugation conditions. In the serum state, miRNAs whose abundance changes depending on the blood collection tube and centrifugation conditions 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 miRNAs with a difference are selected, thereby selecting the reference miRNA. Generally, in DNA microarray measurements, a two-fold change in abundance is considered to be a sufficient difference, so it is preferable to select miRNAs with a two-fold or greater difference between the degraded serum sample and the non-degraded serum sample.

In the measurement process of the present invention, the amount of one or more reference miRNAs selected from miRNAs having the base sequences shown in SEQ ID NOs: 1 to 2 present in a serum sample and a standard serum sample is measured.

Hereinafter, the reference miRNA and the probe for capturing the target miRNA will be collectively referred to as "capture probe" or simply as "probe."

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

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, a chemically modified derivative such as a derivative labeled with a fluorescent group or the like, a derivative containing a modified nucleotide (e.g., a nucleotide containing a halogen, an alkyl group such as methyl, an alkoxy group such as methoxy, a thio group, a carboxymethyl group, or the like, and a nucleotide that has undergone base reconstitution, double bond saturation, deamination, or replacement of an oxygen molecule with a sulfur molecule, etc.).

From the viewpoint of ensuring the stability and specificity of hybridization, it is preferable that the length of the nucleic acid probe is equal to or greater than the length of the miRNA to be detected. Usually, a length of about 17 to 25 bases allows the probe to fully exert selective binding to the target miRNA. Such short oligonucleic acid probes can be easily prepared by well-known chemical synthesis methods.

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 the conditions for obtaining the function of a capture probe prepared for the detection of a desired sample RNA.

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 complementary strand of the reference miRNA shown in SEQ ID NOs: 1 to 2 is used as a probe to measure the abundance of the reference miRNA, the change in the abundance of cross-hybridizing RNA other than the reference miRNA is also detected as a "change in the abundance of the reference miRNA." In the present invention, the quality of a serum sample can be determined by making a judgment that also takes into account the change in the abundance of such cross-hybridizing RNA.

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.

The number of miRNA capture probes immobilized on the support is not particularly limited. For example, the amount of miRNA present may be measured using a support immobilized with a number of miRNA capture probes covering all known miRNAs whose sequences have been identified, or a support immobilized with a desired number of miRNA capture probes depending on the purpose of the test, etc.

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

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

Examples of the former method include the method of Ronald et al. (U.S. Patent No. 5,705,610), the method of Michel et al. (U.S. Patent No. 6,142,266), and the method of Francesco et al. (U.S. Patent No. 7,037,659). In these methods, organic solvents are used during the DNA synthesis reaction, so it is preferable that the support be made of a material that is resistant to organic solvents. In addition, in the method of Francesco et al., DNA synthesis is controlled by irradiating light from the back of the support, so it is preferable that the support be made of a material that is light-transmitting.

Examples of the latter method include the method by Hirota et al. (Patent No. 3922454) and the 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. To immobilize the oligo-DNA on 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 that has an amino group or the like.

Hybridization with each miRNA capture probe immobilized on a support is performed by preparing a nucleic acid sample (nucleic acid sample derived from a specimen) labeled with a labeling substance from RNA extracted from the specimen (specimen RNA) and contacting this labeled nucleic acid sample with the probe. "Nucleic acid sample derived from a specimen" includes not only RNA extracted from the specimen, but also cDNA and cRNA prepared from the RNA by reverse transcription reaction. A 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.

Methods for binding a labeling substance to a nucleic acid sample derived from a specimen include binding a labeling substance to the 3' end of the nucleic acid sample, binding a labeling substance to the 5' end, and incorporating a nucleotide bound to a labeling substance into the nucleic acid. Enzyme reactions can be used for the methods of binding a labeling substance to the 3' end and the 5' end. T4 RNA Ligase, Terminal Deoxitidyl Transferase, Poly A polymerase, etc. can be used for the enzymatic reaction. For both labeling methods, the method described in "miRNA Experimental Protocols, edited by Shao-Yao Ying, Yodosha, 2008" can be used as a reference. 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 (Exicon), NCode miRNA Labeling system (Life Technologies), and FlashTag Biotin RNA Labeling Kit (Genisphere).

In addition, as in conventional methods, 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, which is then hybridized with a probe on an array.

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.

In addition, luminescent semiconductor particles may be used as the labeling substance. Examples of such semiconductor particles include cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), and silver indium zinc sulfide (AgInZnS).

The nucleic acid sample 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 process can be carried out in exactly the same way as in the past. The reaction temperature and time are appropriately selected depending on 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 and 1 minute to 10 hours. After hybridization and washing, the signal intensity from the labeling substance in each probe immobilization area on the support is detected. The signal intensity is detected using an appropriate signal reading device depending on the type of labeling substance. When a fluorescent dye is used as the labeling substance, a fluorescent microscope, a fluorescent scanner, etc. can be used.

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

If the detected measurement value contains background noise, the background noise may be subtracted. The surrounding noise may also be treated as background noise and subtracted from the detected measurement value. Alternatively, the method described in "Microarray Data Statistical Analysis Protocols, edited by Fujibuchi Wataru and Horimoto Katsuhisa, Yodosha, 2008" may be used.

<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.

Conventional methods can be used as the correction method, for example, the global normalization method and the quantile normalization method, which perform correction using the measured values of all detected miRNAs. 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 specific endogenous miRNAs for correction, or using external standard nucleic acids 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 miRNAs contained in serum samples, it is preferable to use a correction method that uses external standard nucleic acids such as spike controls that are not dependent on the sample.

<Comparison process>
The comparison step of the present invention is a step of obtaining a 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 and 2, obtained in the measurement step.

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 and 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 can 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 can 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 can 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 can 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 can 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 can be determined.

<Determination step>
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 the 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.

The quality can be determined by setting a threshold value in advance as a standard for determining the quality of one or more types of reference miRNA contained in a serum sample and a standard serum sample, or the difference or ratio of their index values, and determining whether the quality (good or bad) exceeds the threshold value. 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 determined 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 determined to be good. The determination step in this embodiment can be called a second comparison step in which a comparison with a threshold value is performed.

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 to make the determination. When performing logarithmic transformation, it is common to convert to a logarithm with a base of 2.

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

When multiple reference miRNAs among the miRNAs consisting of 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 calculated, and the quality can be judged based on whether the difference or ratio between these index values exceeds a reference threshold value. Here, the index value can be the sum, average, or median of the abundance of the multiple reference miRNAs.

When multiple reference miRNAs among the 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 for each reference miRNA may be calculated, and whether or not each reference miRNA exceeds a reference threshold value may be determined. In this case, it is preferable to set further judgment criteria by prioritizing or weighting the individual judgments based on the multiple reference miRNAs.

When using one reference miRNA, one miRNA may be arbitrarily selected from the miRNAs shown in SEQ ID NO: 1-2. It is preferable to select one whose abundance is shown to vary particularly sensitively with the addition of leukocytes in the Examples below, but either hsa-miR-6769b-5p (SEQ ID NO: 1) or hsa-miR-6842-5p (SEQ ID NO: 2) can be preferably selected, since both of these abundances vary significantly with the addition of trace amounts 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 an miRNA that can particularly sensitively detect deterioration of serum samples that occurs depending on blood collection conditions in clinical testing.

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.

In addition, for example, when the purpose is gene expression analysis and the target miRNA corresponds to any of the miRNAs shown in SEQ ID NOs: 1 to 2, a reference miRNA can be selected from the miRNAs excluding the target miRNA.

The threshold value used as the criterion for judgment can be set arbitrarily depending on the purpose of the 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 (Equation 3A below).

When one reference miRNA is used, 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.

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 (Equation 1A).

Furthermore, as shown in Equation 2A, the difference (e-E) 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 (e-E) 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.
e－E＞t2 (Equation 2A).

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).

In the case of considering factors that affect the measurement results in the experimental procedure, 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 can be selected whose abundance ratio is 0.90 or more, more preferably 0.96 or more, when compared 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, and 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".

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 Equation 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.

Alternatively, as shown in Formula 5A, the abundance difference (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 difference (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 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 (Formula 4A)
(e-c)/(E-C)>t5 (Equation 5A).

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.

Alternatively, as shown in Equation 7A, the difference in abundance (e-c) 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 difference in abundance (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 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 (Formula 6A)
(e−c)−(E−C)>t7 (Equation 7A).

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. In this case, as shown in Equation 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 Equation 6A is adopted.

Alternatively, the difference in abundance (E-C) between the abundance E of the reference miRNA contained in a standard serum sample and the abundance C of the stable endogenous miRNA may be used as the threshold value t9, in which case, as shown in formula 9A, when the difference in abundance (e-c) between the abundance e of the reference miRNA contained in a serum sample and the abundance c of the stable endogenous miRNA exceeds the threshold value t9, i.e., the difference in abundance (E-C) between the abundance E of the reference miRNA contained in a 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)
e−c＞E−C (=t9) (Equation 9A).

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 calculated, and the difference or ratio between the two index values can be calculated 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 can be 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 can be used instead of the abundance E of the reference miRNA contained in the standard serum sample, and the judgment can be made using formulas 1B to 9B. The index value can be the sum, average, or median of each abundance.
r/R>t1 (Equation 1B)
r－R＞t2 (Equation 2B)
r>R (=t3) (Equation 3B)
(r/c)/(R/C)>t4 (Formula 4B)
(r-c)/(R-C)>t5 (Formula 5B)
(r/c) - (R/C)>t6 (Formula 6B)
(r-c)-(R-C)>t7 (Formula 7B)
r/c>R/C (=t8) (Formula 8B)
r−c＞RC(=t9) (Equation 9B).

Here, in the above formulas 1A to 9A and 1B to 9B, taking into account experimental error etc., thresholds t1 to t9 may be given a certain margin of error α, and 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 threshold t2 a margin of error.

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

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.

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

In addition, for example, when the purpose is gene expression analysis and the target miRNA in the analysis corresponds to any of the miRNAs shown in SEQ ID NOs: 1 to 2, a reference miRNA can be selected from the miRNAs excluding the target miRNA.

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 abundance measurement values 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 sample and the standard serum sample, the abundance measurement values being measured using an RNA sample prepared from a serum sample and a standard RNA sample prepared from a standard serum sample;
The present invention provides 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 types of reference miRNAs between the serum sample and the standard serum sample; and a determination step of determining the quality of the serum sample based on the difference or ratio between the abundance measurement values or index values thereof of one or more types of reference miRNAs obtained in the comparison step (i.e., a program including instructions for causing one or more computers to execute each of the above steps), and a computer-readable recording medium having the program recorded thereon.

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, a measurement value 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 measurement value. In the measurement acquisition step, in addition to the measurement value of the abundance of the reference miRNA, a measurement value 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 measurement value of one or more target miRNAs may be performed simultaneously with the acquisition of the measurement value 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 quality evaluation method 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.

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 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;
and a determination means 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 by the comparison means.

The measured values of the abundance of one or more types of reference miRNA 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 types of reference miRNA, 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 miRNA 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.

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.

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>.

The determination means is a means for carrying out the determination step of the method for evaluating the quality of a serum sample of the present invention. Details are as described in the <Determination step>.

The determination means can be referred to as a quality output means that determines whether the quality of the serum sample is good or bad based on the difference or ratio of 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.

The quality may be determined 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 types of reference miRNA contained in a serum sample and a standard serum sample exceeds a preset threshold value. The determination means in this embodiment may be called a second comparison means for comparing with the threshold value.

The quality assessment device may be incorporated as a quality assessment unit in an 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.

The miRNA expression level analysis device may be configured to execute the expression analysis of one or more target miRNAs described above and output the analysis results when the judgment means obtains a judgment result that the quality is good. 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 judgment result is that the quality is good, the expression analysis result is output with a clear indication that the expression analysis result of the target miRNA is reliable, and when the judgment result is that the quality is poor, the expression analysis result is output with a clear indication that the expression analysis result of the target miRNA is unreliable or low reliable.

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 structure, but also includes a structure that is distributed as multiple modules or libraries, or a structure that achieves its function by working together with a separate program, such as an OS (Operating System). Well-known structures and procedures can be used for the specific structure for reading the recording medium, the reading procedure, or the installation procedure after reading.

A "recording medium" may be any "portable physical medium" (non-transient recording medium) such as a flexible disk, optical magnetic disk, ROM, EPROM, EEPROM, CD-ROM, MO, 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.

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 consisting of the base 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 consisting of the base 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.

The chip of the present invention may further have correction nucleic acids, such as housekeeping RNA, specific endogenous miRNA for correction, and external standard nucleic acids to be added, used in the correction process, and in particular a probe for capturing endogenous miRNA for correction, immobilized on a support.

Below, we will explain publicly known information about 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.

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, as well as other biological species homologs or orthologs. 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 precursor, the "hsa-mir-6769b gene" (miRbase Accession No. MI0022706, SEQ ID NO: 3), which has a hairpin-like structure. The terms "miR-6769b-5p" and "hsa-miR-6769b-5p" also encompass precursors of such hairpin-like structures.

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 nucleotide sequence shown in SEQ ID NO: 2, as well as other biological species homologs or orthologs. It can be obtained by the method described in Ladwig E et al., 2012, Genome Res., vol. 22, pp. 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.

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 explained in more detail below. However, the present invention is not limited to the following examples. In the following examples, the numerical value obtained after 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.

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.

(Preparation of serum samples)
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).

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

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. After hybridization, the DNA microarray 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.

The measured values of each miRNA detected by DNA microarray were converted to logarithms with a base of 2 and corrected by quantile normalization to obtain the expression level of each miRNA.

(Selection of reference miRNAs)
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.

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 to that in serum with 1,000 or 10,000 leukocytes 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, those that were stably detected in the high expression region were narrowed down to obtain the standard miRNAs shown in Table 1. All three miRNAs described in Patent Document 1 (miR-343-3p, miR-150-5p, miR-146a-5p) had low expression and were not selected.

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 using 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 abundance varied by more than 2-fold (more than 1 in terms of the difference in logarithmic base 2 values).

Generally, in DNA microarray measurements, a two-fold change in abundance is considered 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. This confirmed that the miRNA in question can be used as an 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 determined by measuring the abundance of the reference miRNAs shown in Table 1.

Figure 1 shows the abundance of two types of reference miRNAs shown in Table 1 under standard conditions and under conditions in which the amount of white blood cells mixed in was changed (two conditions). The abundance of both hsa-miR-6769b-5p (sequence number 1) and hsa-miR-6842-5p (sequence number 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 more than 1,000 white blood cells, the abundance of hsa-miR-6769b-5p of 5.0 can be set as the threshold, and if the abundance of hsa-miR-6769b-5p in a serum sample exceeds that value, it can be determined that the sample is deteriorated, that is, that the sample is of poor quality.

Based on the results of this Example 1, a specific example of the 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 using the above formula 3A is shown in Table 2 below. In the example of Table 2, the threshold value t3 is set to E±α (α = 0.1 to 0.3). When the abundance e of the reference miRNA contained in the serum sample exceeds the corresponding threshold value, the serum sample can be determined to be of poor quality.

Example 2: Detection of deterioration due to contamination with white blood cells using multiple miRNAs It is also possible to determine deterioration in serum sample quality using a combination of any two types of reference miRNAs, rather than a single miRNA.

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 1,000 or more white blood cells were used. The average abundance of these two types of miRNA was used as the index value for serum samples under each condition. For example, the index value for serum under standard conditions is (4.9 + 5.2)/2 = 5.05.

The abundance of each of these miRNAs under each condition is 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 if 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 quality of the sample is poor. This determination mode corresponds to the above-mentioned formula 3B in which the threshold value t3 is set to R or R ±α.

Example 3
Blood was collected from one healthy individual and serum samples were prepared. The obtained serum was dispensed in 300 μL portions and immediately placed 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 amount of expression. 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 level.

hsa-miR-6769b-5p (SEQ ID NO: 1) was selected as the standard miRNA to be used for quality assessment. 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 determined to be good.

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.

Example 4
The serum sample was changed to one in which 1,000 white blood cells were artificially mixed into the serum sample, 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.

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 contamination with miRNAs derived from leukocytes had occurred, deteriorating the quality. This was consistent with the quality assessment results according to the present invention.

<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 comparing RNA extracted from the serum sample contaminated with 1000 white blood cells 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.

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

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.

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 (11)

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;
the method comprising: a comparing 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 measuring 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 determining step of determining whether or not the serum sample is contaminated by white blood cells based on the difference or ratio in the abundance or index value of the one or more types of reference miRNA obtained in the comparing step. 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. The method according to claim 1 or 2, wherein the measurement 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 abundance of the one or more reference miRNAs in the serum specimen and the standard serum specimen. 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. The method according to any one of claims 1 to 4, wherein the measurement step includes 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. The method according to claim 5, wherein the measurement step includes 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. 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 in the serum sample and the measured values of the abundance of the one or more reference miRNAs obtained in the measurement step. 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 program for carrying out a comparison step of 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; and a determination step of determining whether or not the serum sample is contaminated by white blood cells based on the difference or ratio between the abundance measurement values or index values thereof of one or more types of reference miRNA obtained in the comparison step. A computer-readable recording medium on which the program according to claim 8 is recorded. 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. 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;
and a determination means for determining whether or not the serum sample is contaminated with white blood cells based on the difference or ratio of the abundance measurement values or index values thereof of one or more types of reference miRNA obtained by the comparison means.