WO2018056432A1

WO2018056432A1 - Method for detecting hemolysis in blood sample and hemolysis detection chip

Info

Publication number: WO2018056432A1
Application number: PCT/JP2017/034447
Authority: WO
Inventors: 一恵名取; 近藤　哲司
Original assignee: 東レ株式会社
Priority date: 2016-09-26
Filing date: 2017-09-25
Publication date: 2018-03-29
Also published as: JPWO2018056432A1

Abstract

A new method for detecting hemolysis in a blood sample at a high sensitivity is disclosed. With the hemolysis detecting method according to the present invention, at least one type of miRNA the abundance of which increases in a blood sample in which hemolysis is occurring is used as a hemolysis marker, hemolysis in the blood sample is detected by using the abundance thereof as an indicator, and thus, the presence/absence of hemolysis is assessed. With the method according to the present invention, it is possible to detect hemolysis at a high sensitivity even in a blood sample in which the degree of hemolysis is low and spillage of the contents of red blood cells, such as hemoglobin, is low.

Description

Method for detecting hemolysis of blood sample and chip for hemolysis detection

The present invention relates to a method for detecting hemolysis of a blood sample and evaluating the presence or absence of hemolysis, a chip used for detection of hemolysis, and a chip for miRNA expression analysis.

MiRNA (microRNA) is transcribed from genomic DNA as RNA (precursor) with a hairpin-like structure. This precursor is cleaved by a dsRNA cleaving enzyme (Drosha, Dicer) having a specific enzyme RNase III cleavage activity, then changed to a double-stranded form, and then becomes a single strand. One of the antisense strands is incorporated into a protein complex called RISC and is considered to be involved in mRNA translational suppression. As described above, miRNA has a different form at each stage after transcription.Therefore, when miRNA is targeted for detection, various forms such as a hairpin structure, a double-stranded structure, and a single-stranded structure are usually used. It is necessary to consider. miRNA consists of RNA of 15 to 25 bases, and its existence has been confirmed in various organisms.

In recent years, miRNA is present not only in cells but also in blood samples such as serum and plasma, which are samples that do not contain cells, and their abundance is a biomarker (indicator of various diseases including cancer). ) Is suggested. As of August 2016, more than 2500 miRNAs exist in humans, and when using a highly sensitive measurement system such as a DNA microarray, the abundance of more than 1000 miRNAs can be detected simultaneously in serum and plasma. Is possible. Therefore, biomarker search research targeting serum and plasma using the DNA microarray method has been carried out, and development to biomarker tests that can detect diseases early is expected.

However, it is known that miRNA derived from a hemolyzed blood sample cannot accurately measure the abundance, which may reduce the reliability of the measurement result. As a biomarker of disease, in a test that measures the abundance of miRNA contained in a blood sample, if an examination / diagnosis is performed based on the measurement of the abundance with uncertainty, an appropriate treatment opportunity will be provided. Missing or applying the wrong medical care can put unnecessary financial and physical strain on the patient. Therefore, in order to accurately measure the abundance, it is very important to confirm the presence or absence of hemolysis and use a non-hemolyzed blood sample for the test.

Hemolysis is caused by various factors such as physical, chemical or biological damage to the blood cell membrane of erythrocytes in the blood, causing hemoglobin or miRNA to escape into the serum or plasma. That is. Causes of hemolysis include mechanical stimuli, chemical substances, changes in temperature, heat, osmotic pressure, etc., as well as various direct and indirect effects, including external forces such as vibration and ultrasound on blood samples, electric and magnetic fields, etc. However, the cause of hemolysis is not limited to these. For example, it is known that hemolysis occurs due to the influence of blood collection procedures, and bubbles may enter when sucking blood due to inadequate syringes, or strong vortices may be wound when sucked into a vacuum blood collection tube. May cause hemolysis as a factor. In addition, mixing with an anticoagulant may be caused by shaking vigorously or by giving a strong vibration when transporting a blood sample. Furthermore, it is known that hemolysis occurs even when whole blood is left for a long time without separating serum or plasma.

The degree of hemolysis can be evaluated by measuring the amount of miRNA, hemoglobin, etc. shed from the red blood cells in the blood sample. For example, there is a method of measuring hemoglobin concentration in blood using a spectrophotometer or the like. In addition, color tone comparison with a color sample by visual observation is used as a simple method, which utilizes the fact that hemoglobin is a protein having heme as a red pigment.

In recent years, as miRNA research is actively conducted as described above, there is a concern about the effect of hemolysis on the measurement value. As one example of such research, hsa-miR-451a is used as a hemolysis marker for blood samples. It has been reported to be used as This is a method for evaluating the presence or absence of hemolysis using the difference between the abundance of hsa-miR-451a and the abundance of hsa-miR-23a-3p that is not affected by hemolysis (Non-patent Document 1). On the other hand, it has been reported that hsa-miR-451a is also used as a cancer marker such as gastric cancer (Patent Document 1).

JP 2013-85542 JP

As a biomarker of disease, in a test that measures the abundance of miRNA contained in a blood sample, a slight abundance difference in the target miRNA affects the test result. It is important to detect the presence or absence of hemolysis, evaluate the presence or absence of hemolysis, and use a non-hemolyzed sample. However, in the color comparison with the color sample by the naked eye which is the conventional method, for example, if the hemoglobin concentration is not less than 200 mg / dL, the red color is easily observed with the naked eye unless the specimen has a high degree of hemolysis. It ’s difficult. Samples with a low degree of hemolysis have a slight color difference even when compared with samples with a hemoglobin concentration of 0 mg / dL, and it is difficult to distinguish the presence or absence of hemolysis with the naked eye. Further, the determination result is easily affected by individual differences by the observer and lacks reliability. In addition, the method of measuring blood hemoglobin concentration using a spectrophotometer or the like is preferable because it requires an additional measurement step separate from the measurement of the abundance of miRNA, resulting in increased test costs and process complexity. Absent.

Hsa-miR-451a used as the above-mentioned hemolysis marker has a small variation in the abundance even in a specimen having a hemoglobin concentration of 200 mg / dL as shown in Comparative Example 3 of the present specification, and has low detection sensitivity of hemolysis . In addition, as described above, hsa-miR-451a has been reported to be used as a cancer marker for gastric cancer and the like. It is not preferable to use it as a hemolysis marker.

Thus, in the case of a specimen with a low degree of hemolysis, it is difficult to detect hemolysis with the conventional method, and it is difficult to obtain an accurate gene expression analysis result. The problem to be solved by the present invention is to obtain an accurate gene expression analysis result from a blood sample.For example, even a sample with a low hemolysis concentration of hemoglobin concentration of 100 小さい mg / dL can be easily and highly sensitively. To provide a method for evaluating the presence or absence of hemolysis using miRNA capable of detecting hemolysis as a new hemolysis marker.

As a result of intensive studies to solve the above problems, the present inventors have studied miRNA that can detect hemolysis with high sensitivity, even in a blood sample that has a low degree of hemolysis and a small amount of red blood cell contents such as hemoglobin. As a disease biomarker, a miRNA capable of evaluating the presence or absence of hemolysis at a level that can be judged as appropriate in a test for measuring the abundance of miRNA contained in a blood sample was found, and the present invention was completed.

The present invention comprises the following aspects.
(1) miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073, miR-6131, miR-642b in blood samples A method for detecting hemolysis, comprising a measurement step of measuring the abundance of at least one miRNA selected from the group consisting of -3p, miR-92a-3p and miR-92b-3p.
(2) miR-191-5p is hsa-miR-191-5p, miR-22-3p is hsa-miR-22-3p, miR-3158-5p is hsa-miR-3158-5p MiR-320a is hsa-miR-320a, miR-484 is hsa-miR-484, miR-486-5p is hsa-miR-486-5p, miR-6073 is hsa-miR-6073 MiR-6131 is hsa-miR-6131, miR-642b-3p is hsa-miR-642b-3p, miR-92a-3p is hsa-miR-92a-3p, miR-92b The method according to (1), wherein -3p is hsa-miR-92b-3p.
(3) The method for detecting hemolysis according to (1) or (2), wherein the miRNA is a polynucleotide shown in any of the following (a) to (e):
(a) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1 to 11 or a base sequence in which u is t in the base sequence;
(b) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1 to 11 or a base sequence in which u is t in the base sequence.
(c) a polynucleotide comprising a base sequence represented by any one of SEQ ID NOs: 1 to 11 or a base sequence complementary to a base sequence in which u is t in the base sequence.
(d) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1 to 11 or a base sequence complementary to the base sequence in which u is t in the base sequence;
(e) a polynucleotide that hybridizes with the polynucleotide of any one of (a) to (d) under stringent conditions.
(4) Any one of (1) to (3), further comprising a correction step of correcting the measurement value of the abundance of the miRNA contained in the blood sample and obtaining the corrected measurement value as the expression level of the miRNA. The method according to item.
(5) The abundance or expression level of the miRNA in the blood sample, the difference between the abundance or expression level and the abundance of the standard endogenous miRNA or external standard nucleic acid in the blood sample, or the abundance or expression level When the ratio of the standard endogenous miRNA or the external standard nucleic acid in the blood sample exceeds a threshold value that is predetermined as a reference for evaluating the presence or absence of hemolysis, the blood sample is evaluated as a hemolytic sample The method according to any one of (1) to (4), comprising an evaluation step.
(6) The measurement step contacts a probe for capturing at least one miRNA selected from the miRNA immobilized on a support and a nucleic acid sample extracted from a blood sample and labeled with a labeling substance. The method according to any one of (1) to (5), wherein hybridization is performed to measure the abundance of the miRNA contained in the blood sample.
(7) The measurement step includes measuring the abundance of a desired target miRNA contained in the blood sample simultaneously with measurement of the abundance of at least one miRNA selected from the miRNA contained in the blood sample. The method according to any one of (1) to (6).
(8) miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073 miRNAs for detecting hemolysis in blood samples , MiR-6131, miR-642b-3p, miR-92a-3p and miR-92b-3p, comprising a support on which is immobilized a probe for capturing at least one miRNA selected from the group consisting of miR-92b-3p For chips.
(9) miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073 which are miRNAs for detecting hemolysis in blood samples A probe for capturing at least one miRNA selected from the group consisting of miR-6131, miR-642b-3p, miR-92a-3p and miR-92b-3p, and a probe for capturing a desired target miRNA A chip for miRNA expression analysis comprising an immobilized support.

According to the present invention, it is possible to detect hemolysis with high sensitivity even in a blood sample with a low degree of hemolysis, that is, a blood sample in which hemoglobin or miRNA that is the contents of red blood cells is small, which has been difficult with the conventional method. The presence or absence of hemolysis can be easily and highly sensitively evaluated. In particular, hemolysis can be detected even if the hemoglobin concentration is about 100 mg / dL, so that a slight abundance difference in the target miRNA affects the results, for example, blood as a biomarker for disease. Tests that measure the abundance of miRNA contained in a sample makes it possible to determine the suitability of a blood sample (select a blood sample that is not affected by hemolysis and provide it for testing), and provide highly reliable test results Can be obtained.

The present invention is a method for detecting hemolysis of a blood sample, miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR- In this method, at least one miRNA selected from the group consisting of 6073, miR-6131, miR-642b-3p, miR-92a-3p and miR-92b-3p is used as a “hemolysis marker”. Here, in this specification, miRNA that serves as an index for detecting the degree or presence of hemolysis in a blood sample may be referred to as a “hemolysis marker”.

“MiRNA” is a kind of non-coding RNA (ncRNA) that is a short-chain RNA of about 15 to 25 bases produced in vivo, and is considered to have a 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 dsRNA cleaving enzyme (Drosha, Dicer) having a specific enzyme RNase III cleavage activity, then changed to a double-stranded form, and then becomes a single strand. One of the antisense strands is incorporated into a protein complex called RISC and is considered to be involved in mRNA translational suppression. As described above, miRNA has different aspects at each stage after transcription. Therefore, when miRNA is targeted (detection target), normally, such as a hairpin structure, a double-stranded structure, a single-stranded structure, etc. Various forms need to be considered. The presence of miRNA has been confirmed in various organisms.

Examples of animal species from which blood samples to which the present invention can be applied include mammals such as humans, mice, rats, dogs, pigs, monkeys, hamsters, guinea pigs, cows, horses, rabbits, sheep, goats, cats, etc. Although not limited to these, human is preferable.

The blood sample to which the present invention can be applied is a sample separated from a living body, and examples thereof include serum and plasma.

In the present invention, RNA can be extracted from these blood samples, and the abundance of miRNA can be measured using this RNA. RNA extraction is a known method (for example, the method of Favaloro et al. (Favaloro et.al., Methods Enzymol. 65: 718 (1980))), and various kits therefor are commercially available (for example, Qiagen miRNeasy, "3D-Gene" (RNA) extraction (reagent) from liquid (sample) etc. from Toray Industries, Inc. can be applied.

<Measurement process>
The method of the present invention comprises miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073, miR-6131 contained in a blood sample. Measuring the abundance of at least one miRNA selected from the group consisting of miR-642b-3p, miR-92a-3p and miR-92b-3p. Further, as will be described later, simultaneously with the measurement of the abundance of the miRNA contained in the blood sample, the abundance of a desired target miRNA such as a disease biomarker or the abundance of a correction standard nucleic acid may be measured.

Generally, when hemolysis occurs, the contents of red blood cells flow out into the serum and plasma, so that the amount of nucleic acid containing miRNA in the blood sample increases. The miRNA used as a hemolysis marker of the present invention is a miRNA that is selected as a miRNA whose abundance increases in a hemolyzed specimen, and whose abundance increases well in correlation with the degree of hemolysis, that is, the hemoglobin concentration. As a method for selecting a hemolysis marker, for example, in the expression level obtained by correction described below, the expression level ratio between the specimen having a hemoglobin concentration of 0 mg / dL and the specimen having a hemoglobin concentration of 40 mg / dL is preferably 2 or more, It is preferable to select miRNAs that are more preferably 3 or more. In addition, by applying a statistical technique, miRNAs whose increase in abundance is statistically significant in hemolyzed specimens can be selected by comparison between groups in gene expression analysis. For example, a statistical analysis method based on a commonly used t test may be used. For example, the statistical language “R” package “SAM” (Tusher VG et. Al., Proc Natl Acad Sic USA. 2001 98 (9) 5116-5121) can be applied as it is.

As described above, since the contents of miRNA in red blood cells flow into serum and plasma when hemolysis occurs, RNA obtained from hemolyzed specimens (hereinafter also referred to as “hemolyzed specimens”) and non-hemolyzed specimens ( Hereinafter, when compared with RNA obtained from “non-hemolyzed specimen”), there is a difference in the abundance of various miRNAs contained therein. Therefore, in a gene expression analysis, if a hemolyzed sample is included, the correlation between the samples decreases, and the abundance of the target miRNA in the blood sample cannot be accurately measured.

In gene expression analysis, when the abundance ratio (fluctuation in abundance) of target biomarkers is 2 times or more, the difference is usually judged to be significant. In other words, if there is hemolysis to the extent that the abundance ratio of the target biomarker is twice or more, the result of the gene expression analysis will be unreliable. For example, when miRNA such as Let-7 series, which is known as a cancer marker, is used as a target biomarker, results obtained from a hemolyzed sample with a hemoglobin concentration of 100 mg / dL and non-hemolyzed hemoglobin with a hemoglobin concentration of 0 mg / dL When compared with results obtained from specimens, abundance ratios of more than twice may occur. As a result, the test result is inaccurate and unreliable, such as a specimen that should originally be determined to be non-cancerous is determined as cancer. Thus, in a test where a slight difference in the amount of target biomarkers such as gene expression analysis affects the test result, a hemoglobin sample with a hemoglobin concentration of 100 mg / dL is a hemolyzed sample. It is important that it can be detected as

The method of the present invention is accurate in gene expression analysis for measuring the abundance of a gene product contained in a specimen, for example, analysis using an array chip such as a microarray, analysis by polymerase chain reaction (PCR) method, or sequencing method. It can be used to obtain analysis results. In gene expression analysis, for example, a miRNA contained in a blood sample is labeled and a probe for capturing one or a plurality of target miRNAs and a probe for capturing a miRNA of a hemolytic marker are immobilized on a support. Contacting the labeled miRNA and capturing the miRNA with the probe, measuring the abundance of various miRNAs, a probe for amplifying one or more target miRNAs, and a probe for amplifying the miRNA of the hemolytic marker To measure the abundance of the target miRNA, and to use these results to analyze and test gene expression, for example, to measure clinical specimens to understand the pathology To perform an inspection.

Hereinafter, probes for capturing nucleic acids, such as hemolytic markers, target miRNAs described later, and standard nucleic acids for correction, are also collectively referred to as “capture probes” or simply “probes”. In particular, probes for capturing miRNAs are “ Also referred to as “miRNA capture probe”.

Measurement of the abundance of miRNA can be performed, for example, by a hybridization assay using an array chip such as a microarray in 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 one or a plurality of “hemolytic marker capture probes” for capturing miRNA for detecting hemolysis is immobilized can be used. In addition, by using an array chip including a support on which a “target miRNA capture probe” for capturing a target miRNA described later and a “standard nucleic acid capture probe for correction” for capturing a standard nucleic acid for correction are further immobilized. Also good.

“Capture probe” or “probe for capturing” means a substance capable of binding directly or indirectly, preferably directly and selectively to the miRNA to be captured. , Nucleic acids, proteins, saccharides and other antigenic compounds. In the present invention, a nucleic acid probe can be preferably used. In addition to DNA and RNA, nucleic acid derivatives such as PNA (peptide nucleic acid) and LNA (LockedLNucleic ほか Acid) can be used as the nucleic acid. Here, in the case of nucleic acid, a derivative means a labeled derivative with a fluorophore, a modified nucleotide (for example, a nucleotide containing a group such as halogen, alkyl such as methyl, alkoxy such as methoxy, thio, carboxymethyl, etc.) Means a chemically modified derivative such as a derivative comprising a nucleotide having undergone a constitution, saturation of a double bond, deamination, substitution of an oxygen molecule with a sulfur molecule, and the like.

The chain length of the nucleic acid probe is preferably not less than the length of the miRNA to be detected from the viewpoint of ensuring the stability and specificity of hybridization. Usually, if the chain length is about 17 to 25 bases, the probe can sufficiently exhibit selective binding to the target miRNA. Such an oligonucleic acid probe having a short chain length can be easily prepared by a known chemical synthesis method or the like.

It is preferable that the nucleic acid probe has a base sequence that is completely complementary to the target miRNA. However, even if there are some differences, the base is highly homologous enough to hybridize with the target miRNA under stringent conditions. Any array can be used as a capture probe.

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. For example, the conditions described in Sambrook, J. et al. (1998) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York can be used. . Stringent temperature conditions are about 30 ° C or higher. Other conditions include the hybridization time, the concentration of the detergent (eg, SDS), 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 a function as a capture probe prepared for detection of a desired sample RNA.

miRNA sequence information can be obtained from databases such as GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and miRBase website (http://www.mirbase.org/). it can. A hemolytic marker capture probe, a target miRNA capture probe, and a standard nucleic acid capture probe for correction can be designed based on sequence information available from these sites.

The number of miRNA capture probes immobilized on the support is not particularly limited. For example, the abundance of miRNA may be measured using a fixed number of miRNA capture probes covering all known miRNAs whose sequences have been identified on a support, or for testing purposes, etc. Accordingly, a desired number of miRNA capture probes immobilized on a support may be used.

As the support on which the capture probes are aligned and fixed, the same support as that used in known microarrays and macroarrays can be used. For example, a slide glass, a membrane, beads or the like can be used. it can. A support having a shape having a plurality of convex portions on the surface described in Japanese Patent No. 4244788 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; polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and silicone rubber.

As a method for immobilizing a capture probe on a support, there are known a method of synthesizing oligo DNA on the surface of the support and a method of dropping oligo DNA synthesized in advance on the surface of the support and immobilizing it.

Examples of the former method include the method of Ronald et al. (US Pat. No. 5,705,610), the method of Michel et al. (US Pat. No. 6,142,266), and the method of Francesco et al. (US Pat. No. 7037659). . In these methods, since an organic solvent is used during the DNA synthesis reaction, the support is preferably made of a material resistant to the organic solvent. In the method of Francesco et al., DNA synthesis is controlled by irradiating light from the back surface of the support, and therefore the support is preferably made of a light-transmitting material.

Examples of the latter method include the method of Hirota et al. (Patent No. 3922454) and a method using a spotter. Examples of the spot method include a pin method based on mechanical contact of a pin tip with a solid phase, an ink jet method utilizing the principle of an ink jet printer, and a capillary method using a capillary tube. After spot treatment, post-treatment such as cross-linking by UV irradiation and surface blocking is performed as necessary. In order to immobilize the oligo DNA by covalent bonding to the surface of the surface-treated support, a functional group such as an amino group or an SH group is introduced at the end of the oligo DNA. The surface modification of the support is usually performed by treatment with a silane coupling agent having an amino group or the like.

For hybridization with various miRNA capture probes immobilized on a support, a nucleic acid sample labeled with a labeling substance (sample-derived nucleic acid sample) is prepared from RNA extracted from the sample, and the labeled nucleic acid sample is probed. It is carried out by contacting with. The “sample-derived nucleic acid sample” includes not only RNA extracted from the sample, but also cDNA and cRNA prepared from the RNA by a reverse transcription reaction. The labeled sample-derived nucleic acid sample may be a sample obtained by directly or indirectly labeling a sample RNA with a labeling substance, or a cDNA or cRNA prepared from the sample RNA may be directly or indirectly labeled with a labeling substance. It may be labeled.

Methods for binding a labeling substance to a sample-derived nucleic acid sample 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 the labeling substance into the nucleic acid. Can be mentioned. An enzymatic reaction can be used in the method of binding a labeling substance to the 3 ′ end and the method of binding a labeling substance to the 5 ′ end. For the enzyme reaction, T4-RNA Ligase, Terminal Deoxitidil Transferase, Poly A-polymerase, etc. can be used. Any labeling method can be referred to the method described in “Shao-YaoYYing, edited by miRNA experiment protocol, Yodosha, 2008”. Various kits for binding a labeling substance directly or indirectly to the end of RNA are commercially available. For example, as a kit for binding a labeling substance directly or indirectly to the 3 ′ end, “3D-Gene” miRNA labeling kit (Toray), miRCURYmimiRNA HyPower labeling kit (Exicon), NCode miRNA Labeling system (Life Technologies Co., Ltd.), FlashTag Biotin RNA Labeling Kit (Genisphere), and the like.

In addition, as in the conventional method, cDNA or cRNA incorporating a labeled substance is prepared by synthesizing cDNA or cRNA from sample RNA in the presence of labeled deoxyribonucleotides or labeled ribonucleotides, and this is arrayed. A method of hybridizing with the above probe is also possible.

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

Further, as the labeling substance, semiconductor fine particles having a light emitting property may be used. Examples of such semiconductor fine particles include cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), silver indium zinc sulfide (AgInZnS), and the like.

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 performed in 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 several tens of hours. Hybridization is performed, and after washing, the signal intensity from the labeling substance in each probe-immobilized region on the support is detected. The signal intensity is detected using an appropriate signal reader according to the type of labeling substance. When a fluorescent dye is used as a labeling substance, a fluorescence microscope or a fluorescence scanner may be used.

Measured value of detected fluorescence intensity is compared with ambient noise. Specifically, the measured value obtained from the probe-immobilized region is compared with the measured value obtained from other positions, and the case where the former numerical value is exceeded is detected (effective determination positive). .

If the detected measurement value includes background noise, the background noise may be subtracted. The ambient noise can be subtracted from the detected measurement value as background noise. In addition, you may use the method described in "Fujitoko, Katsuhisa Horimoto edition, microarray data statistical analysis protocol, Yodosha, 2008".

<Correction process>
As the abundance of the hemolytic marker obtained in the measurement step, the measurement value may be used as it is, but the measurement value is corrected using a standard nucleic acid for correction, and the corrected measurement value is obtained as the expression level of the hemolysis marker. The expression level may be used to evaluate hemolysis. In particular, when hemolysis is to be detected with high sensitivity for the purpose of gene analysis or the like, it is preferable to evaluate using the expression level obtained by correcting the measured value using the standard nucleic acid for correction.

Standard nucleic acids for correction include housekeeping RNAs such as U1snoRNA, U2snoRNA, U3snoRNA, U4snoRNA, U5snoRNA, U6snoRNA, 5S rRNA, and 5.8S rRNA (see JP 2007-75095 A; JP 2007-97429 A). Endogenous miRNA for correction of (Roberts, TC et al., 2014, PLoS ONE, 9 (2), e89237; Chen, X. et al., 2013, PLoS ONE, 8 (11), e79652; WO 2016 / 043170 or the like) or by using an external standard nucleic acid added during RNA extraction or labeling. “Endogenous” means not naturally added to the specimen, but naturally present in the specimen. For example, “endogenous miRNA” refers to miRNA that is naturally present in the sample and is derived from the organism that provided the sample, hsa-miR-149-3p (SEQ ID NO: 12), hsa-miR -2861 (SEQ ID NO: 13), hsa-miR-4463 (SEQ ID NO: 14) and the like are included. These endogenous miRNAs are known to remain in abundance even in hemolyzed specimens, and gene expression analysis of desired target miRNAs such as disease biomarkers contained in blood specimens is performed by applying the method of the present invention. In such a case, it is preferable to use a method of correcting the measured value of the abundance to a constant value using endogenous miRNA having a constant abundance. In the present specification, an endogenous miRNA whose abundance in a blood sample is constant regardless of the presence or absence of hemolysis may be referred to as a “standard endogenous miRNA”. Alternatively, a method of correcting the measured value of the abundance to a constant value using an external standard nucleic acid such as spike control that does not depend on the specimen may be used. For example, the expression level E of the hemolytic marker is a constant value of the measured value a of the abundance of miRNA (standard endogenous miRNA) whose abundance is known to remain unchanged even in hemolyzed samples, as shown in Equation 1. Using the correction coefficient (A / a) for correcting to A, the measurement value e of the abundance of the hemolytic marker can be corrected and acquired.
E = e x A / a (Formula 1)

<Evaluation process>
In the method of the present invention, a threshold value used as a reference for evaluating the presence or absence of hemolysis may be set in advance for the amount of hemolysis marker to be used. The threshold is set for each hemolysis marker. When the measured abundance of the hemolytic marker exceeds a predetermined threshold value, it can be evaluated that the specimen is a hemolytic specimen. When performing a stricter or high-precision evaluation, it is preferable to use the expression level obtained in the above-described correction step as the abundance of the hemolytic marker.

When using one hemolysis marker, miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073, miR-6131, One miRNA may be arbitrarily selected from miR-642b-3p, miR-92a-3p and miR-92b-3p, but the abundance increases significantly depending on the degree of hemolysis, that is, the hemoglobin concentration. For example, it is preferable to select one of miR-486-5p and miR-92a-3p. Moreover, when performing stricter or highly accurate evaluation, it is preferable to use a plurality of hemolytic markers. For example, it is more preferable to use 2 to 5 hemolysis markers, and it is particularly preferable to select two of miR-486-5p and miR-92a-3p. In addition, for example, in the case of gene expression analysis, and when the target miRNA targeted for expression analysis corresponds to any of the hemolysis markers of the present invention, the hemolysis marker is selected from miRNAs excluding the target miRNA. That's fine.

When using one miRNA from the hemolysis marker of the present invention, whether or not this value exceeds a predetermined threshold value using the abundance of the hemolysis marker contained in the blood sample or the expression level thereof The presence or absence of hemolysis can be evaluated. When a plurality of miRNAs are used from among the hemolytic markers of the present invention, it may be evaluated whether or not a threshold value is exceeded for each individual hemolytic marker. In this case, it is preferable to provide further judgment criteria by prioritizing or weighting individual evaluations using a plurality of hemolytic markers.

Threshold values for evaluating the presence or absence of hemolysis can be arbitrarily set according to the purpose of evaluation and the accuracy required. For example, a commercially available blood sample may be used as a standard sample, and the abundance of a hemolysis marker in the standard sample may be used as a threshold for the abundance of the hemolysis marker. Further, the relationship between the hemoglobin concentration of the blood sample and the abundance of the hemolytic marker is measured, and the abundance of the hemolytic marker or a value obtained by multiplying the abundance by an appropriate ratio (for example, 1 to 1.2 times the abundance) A threshold value may be set for (value). In addition, a threshold value may be set for the difference or ratio between the amount of endogenous miRNA or the amount of external standard nucleic acid added during RNA extraction or labeling and the amount of hemolytic marker of the present invention. Furthermore, for each hemolytic marker, the variation (standard deviation SD) between individuals of the abundance may be measured in advance, and the value of this variation may be used as a threshold for the abundance of the hemolysis marker, For example, a median value + 2SD may be set as the upper limit 95% reliability reference value. When measuring the variation between individuals, it is desirable to obtain blood samples from as many individuals as possible, for example, about several tens to several hundreds of individuals. As described above, the threshold value can be arbitrarily set according to the measurement or examination using the blood sample, and the detection sensitivity of hemolysis can be increased by setting the threshold more strictly.

When a threshold value used as a reference for evaluating the presence or absence of hemolysis is set in advance with respect to the abundance of the hemolysis marker contained in the blood sample, it can be evaluated using the abundance measurement value e or expression level E. . When the measurement value e is used, as shown in Formula 2A, when the measurement value e exceeds the threshold value t1, it can be evaluated that the blood sample is a hemolysis sample. When evaluating using the expression level E, as shown in Formula 2B, when the expression level E exceeds the threshold value t2, it can be evaluated that the blood sample is a hemolyzed sample. The threshold values t1 and t2 can be arbitrarily set for each hemolysis marker according to the purpose of evaluation and the accuracy required.
e> t1 (Formula 2A)
E> t2 (Formula 2B)

In addition, the presence of hemolytic markers and endogenous miRNAs (standard endogenous miRNAs) that are known to remain unchanged even in hemolyzed samples, external standard nucleic acids added during RNA extraction and labeling, etc. A threshold value for evaluating the presence or absence of hemolysis with respect to the difference from the amount of nucleic acid may be set in advance, and the presence or absence of hemolysis may be evaluated depending on whether or not the difference exceeds the threshold value. When a plurality of standard endogenous miRNAs or external standard nucleic acids are used, a threshold value may be arbitrarily determined for each combination of miRNA used as a hemolysis marker and standard endogenous miRNA or external standard nucleic acid. When the abundance measurement value e is used as the abundance of the hemolysis marker contained in the blood sample, the difference from the measurement value a of the abundance of standard endogenous miRNA or external standard nucleic acid in the blood sample as shown in Formula 3A (E−a) is obtained, and when the absolute value of this value exceeds the threshold value t3, it can be evaluated that the blood sample is a hemolyzed sample. When the expression level E is used as the abundance of the hemolysis marker contained in the blood sample, as shown in Formula 3B, the abundance A in the blood sample of the standard endogenous miRNA or external standard nucleic acid (becomes a constant value) A difference (E−A) is obtained, and when the absolute value of this value exceeds the threshold value t4, it can be evaluated that the blood sample is a hemolyzed sample.
｜ e−a ｜＞ t3 (Formula 3A)
｜ EA－＞ t4 (Formula 3B)

Alternatively, a threshold for evaluating the presence or absence of hemolysis is set in advance with respect to the ratio between the abundance of the hemolysis marker and the abundance of standard endogenous miRNA or external standard nucleic acid, and whether or not the ratio exceeds the threshold The presence or absence of hemolysis may be evaluated by When a plurality of standard endogenous miRNAs or external standard nucleic acids are used, a threshold value may be arbitrarily determined for each combination of miRNA used as a hemolysis marker and standard endogenous miRNA or external standard nucleic acid. When the measurement value e of the abundance of the hemolytic marker contained in the blood sample is used, as shown in Formula 4A, the ratio (e /?) To the measurement value a of the abundance of the standard endogenous miRNA or external standard nucleic acid in the blood sample a) is obtained, and when this value exceeds the threshold value t5, it can be evaluated that the blood sample is a hemolyzed sample. When the expression level E is used as the abundance of the hemolytic marker contained in the blood sample, as shown in Formula 4B, the abundance A (which is a constant value) in the blood sample of the standard endogenous miRNA or external standard nucleic acid The ratio (E / A) is obtained, and when this value exceeds the threshold value t6, it can be evaluated that the blood sample is a hemolyzed sample.
e / a> t5 (Formula 4A)
E / A> t6 (Formula 4B)

Here, in the above formulas 2A to 4B, in consideration of experimental errors, etc., the thresholds t1 to t6 are given a certain error α width, and “t1 ± α” to “t6 ± α”, respectively. Also good. In this case, the error α may be set arbitrarily. For example, in Equation 2A, about 10% of e can be set as α so that the threshold value t1 has a width.

Also, as a threshold for each abundance, a value obtained by converting the abundance value into a logarithm may be used. In this case, an appropriate threshold value may be set according to the conversion. For example, when Expression 4A is applied, the abundance ratio (e / a) may be converted into a logarithmic value, and the threshold value t5 may be set in accordance with the conversion. In this case, as a result, the difference between the logarithmic values of the abundances e and a is obtained.

Further, the presence or absence of hemolysis can be evaluated according to the evaluation criteria using the abundance contained in the blood sample for each hemolysis marker, and the results can be combined to determine the presence or absence of hemolysis in the blood sample. Specifically, for example, in the determination for each hemolytic marker, when the number of hemolytic markers that have detected hemolysis exceeds the number of hemolytic markers that have not detected hemolysis or any predetermined number, the blood sample is a hemolytic sample. It can be evaluated that there is. Further, in the case of performing stricter or high-precision evaluation, a blood sample may be evaluated as a hemolytic sample when hemolysis is detected with a specific one type of hemolytic marker.

The present invention is miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR- which are miRNAs for detecting hemolysis of a blood sample Hemolysis comprising a support on which a probe for capturing at least one miRNA selected from the group consisting of 6073, miR-6131, miR-642b-3p, miR-92a-3p and miR-92b-3p is immobilized A detection chip is provided. The present invention also includes a probe for capturing a target miRNA, and miR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, which are miRNAs for detecting hemolysis in a blood sample. for capturing at least one miRNA selected from the group consisting of miR-484, miR-486-5p, miR-6073, miR-6131, miR-642b-3p, miR-92a-3p and miR-92b-3p Provided is a chip for miRNA expression analysis comprising a support on which a probe is immobilized. Here, the target miRNA, miRNA (hemolysis marker) for detecting hemolysis, the probe for capturing these, and the support on which these capture probes are immobilized are as described above.

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

In the present invention, miRNAs consisting of the nucleotide sequences represented by SEQ ID NOS: 1 to 11 described below are used as miRNAs (hemolysis markers) for detecting the degree or presence of hemolysis in a blood sample.

As used herein, the term “hsa-miR-191-5p gene” or “hsa-miR-191-5p” refers to the hsa-miR-191-5p gene consisting of the base sequence represented by SEQ ID NO: 1 ( miRBase Accession No. MIMAT0000440) and other species homologs or orthologs. The hsa-miR-191-5p gene can be obtained by the method described in Lagos-Quintana M et al., 2003, RNA, Vol. 9, p175-179. “Hsa-miR-191-5p” includes the sequence of “hsa-mir-191” (miRBasemiAccession No. MI0000465, SEQ ID NO: 15) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-22-3p gene” or “hsa-miR-22-3p” refers to the hsa-miR-22-3p gene consisting of the base sequence represented by SEQ ID NO: 2 ( miRBase Accession No. MIMAT0000077) and other species homologs or orthologs. The hsa-miR-22-3p gene can be obtained by the method described in Lagos-Quintana M et al., 2001, Science, 294, p853-858. “Hsa-miR-22-3p” includes the sequence of “hsa-mir-22” (miRBasemiAccession No. MI0000078, SEQ ID NO: 16) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-3158-5p gene” or “hsa-miR-3158-5p” refers to the hsa-miR-3158-5p gene consisting of the base sequence represented by SEQ ID NO: 3 ( miRBase Accession No. MIMAT0019211) and other species homologs or orthologs. The hsa-miR-3158-5p gene can be obtained by the method described in Creighton CJ et al., 2010, PLoS One, Volume 5. “Hsa-miR-3158-5p” has a hairpin-like structure as its precursor, “hsa-mir-3158-1, hsa-mir-3158-2” (miRBase Accession No. MI0014186, MI0014187, SEQ ID NO: 17 and 18).

As used herein, the term “hsa-miR-320a gene” or “hsa-miR-320a” refers to the hsa-miR-320a gene (miRBase Accession No. MIMAT0000510) consisting of the base sequence shown in SEQ ID NO: 4. And other species homologues or orthologues. The hsa-miR-320a gene can be obtained by the method described in Michael MZ et al., 2003, Mol Cancer Res, Vol. 1, p882-891. In addition, “hsa-miR-320a” includes the sequence of “hsa-mir-320a” (miRBase Accession No. MI0000542, SEQ ID NO: 19) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-484 gene” or “hsa-miR-484” refers to the hsa-miR-484 gene consisting of the base sequence represented by SEQ ID NO: 5 (miRBase から Accession 塩基 No. MIMAT0002174) And other species homologues or orthologues. The hsa-miR-484 gene can be obtained by the method described in Fu H et al., 2005, FEBS Lett, 579, p3849-3854. In addition, “hsa-miR-484” includes the sequence of “hsa-mir-484” (miRBase Accession No. MI0002468, SEQ ID NO: 20) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-486-5p gene” or “hsa-miR-486-5p” refers to the hsa-miR-486-5p gene consisting of the base sequence represented by SEQ ID NO: 6 ( miRBase Accession No. MIMAT0002177) and other species homologs or orthologs. The hsa-miR-486-5p gene can be obtained by the method described in Fu H et al., 2005, FEBS Lett, 579, p3849-3854. “Hsa-miR-486-5p” has a hairpin-like structure as a precursor thereof, “hsa-mir-486-1, hsa-mir-486-2” (miRBaseMIAccession 番号 No. MI0002470, MI0023622, SEQ ID NO: 21, 22).

As used herein, the term “hsa-miR-6073 gene” or “hsa-miR-6073” refers to the hsa-miR-6073 gene consisting of the base sequence represented by SEQ ID NO: 7 (miRBase Accession 配列 No. MIMAT0023698). And other species homologues or orthologues. The hsa-miR-6073 gene can be obtained by the method described in Voellenkle C et al., 2012, RNA, 18, p472-484. “Hsa-miR-6073” includes a sequence of “hsa-mir-6073” (miRBase Accession No. MI0020350, SEQ ID NO: 23) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-6131 gene” or “hsa-miR-6131” refers to the hsa-miR-6131 gene consisting of the base sequence represented by SEQ ID NO: 8 (miRBase Accession 塩基 No. MIMAT0024615). And other species homologues or orthologues. The hsa-miR-6131 gene can be obtained by the method described in Dannemann M et al., 2012, Genome Biol Evol, vol. 4, p552-564. “Hsa-miR-6131” includes the sequence of “hsa-mir-6131” (miRBase Accession No. MI0021276, SEQ ID NO: 24) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-642b-3p gene” or “hsa-miR-642b-3p” refers to the hsa-miR-642b-3p gene comprising the nucleotide sequence represented by SEQ ID NO: 9 ( miRBase Accession No. MIMAT0018444) and other species homologs or orthologs. The hsa-miR-642b-3p gene can be obtained by the method described in Witten D et al., 2010, BMC Biol, Vol. 8, p58. In addition, “hsa-miR-642b-3p” includes the sequence of “hsa-mir-642b” (miRBase Accession No. MI0016685, SEQ ID NO: 25) having a hairpin-like structure as a precursor.

As used herein, the term “hsa-miR-92a-3p gene” or “hsa-miR-92a-3p” refers to the hsa-miR-92a-3p gene consisting of the base sequence represented by SEQ ID NO: 10 ( miRBase Accession No. MIMAT0000092) and other species homologs or orthologs. The hsa-miR-92a-3p gene can be obtained by the method described in Mourelatos Z et al., 2002, Genes Dev, 16, p720-728. “Hsa-miR-92a-3p” has a hairpin-like structure as a precursor thereof, “hsa-mir-92a-1, hsa-mir-92a-2” (miRBase Accession No. MI0000093, MI0000094, SEQ ID NO: 26, 27).

As used herein, the term “hsa-miR-92b-3p gene” or “hsa-miR-92b-3p” refers to the hsa-miR-92b-3p gene consisting of the base sequence represented by SEQ ID NO: 11 ( miRBase Accession No. MIMAT0003218) and other species homologs or orthologs. The hsa-miR-92b-3p gene can be obtained by the method described in Cummins JM et al., 2006, Proc Natl Acad Sci U S A, 103, p3687-3692. “Hsa-miR-92b-3p” includes the sequence of “hsa-mir-92b” (miRBasemiAccession No. MI0003560, SEQ ID NO: 28) having a hairpin-like structure as a precursor.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

<Example 1> Selection of hemolytic marker (DNA microarray)
The following experiment was performed using "3D-Gene" human miRNA oligo chip (compatible with miRBase release 21) manufactured by Toray Industries, Inc.

(Preparation of serum sample)
Blood was collected from healthy humans under two conditions: when sucked vigorously into a vacuum blood collection tube and when slowly sucked into the vacuum blood collection tube, and serum was prepared respectively. The serum sample that was vigorously aspirated into the vacuum blood collection tube was reddish, and as a result of measurement with Eightcheck-3WP (Sysmex Corporation), the hemoglobin concentration was 200 mg / dL and was a hemolyzed sample. On the other hand, the hemoglobin concentration of the serum sample slowly sucked into the vacuum blood collection tube was 0 mg / dL, which was a non-hemolyzed sample. These two serum samples were mixed to prepare blood samples adjusted to have hemoglobin concentrations of 0, 40, 80, 100, 200 mg / dL and a total amount of 400 μL. The obtained serum specimen was stored in a freezer set at −80 ° C. and left as it was until the RNA extraction operation described below.

(Preparation of sample RNA and measurement of miRNA expression level)
The serum sample prepared as described above is dissolved simultaneously, and RNA contained in the serum sample (hereinafter referred to as “sample RNA”) is extracted using “3D-Gene” RNA extraction reagent from liquid sample kit (Toray Industries, Inc.). did.

The obtained sample RNA was labeled using “3D-Gene” “miRNA” labeling kit (Toray Industries, Inc.). The labeled sample RNA was hybridized using “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.), and the fluorescence intensity was measured to obtain the abundance of various miRNAs. The scanner was set to 100% laser output, and the voltage setting of the photomultiplier was set to AUTO.

The abundance of various miRNAs detected by the DNA microarray was corrected using the abundance of the endogenous miRNA hsa-miR-149-3p (SEQ ID NO: 12) to obtain the expression levels of the various miRNAs.

As described above, the expression level was obtained for each serum specimen having hemoglobin concentrations of 0, 40, 80, 100, and 200 mg / dL after detection with a DNA microarray.

(Selection of hemolysis marker)
The miRNA expression levels of the serum samples obtained as described above were compared, and miRNAs whose expression levels varied depending on the degree of hemolysis were selected as hemolysis markers as follows.

First, the ratio between the expression level of each miRNA and the expression level of each miRNA in each serum sample with a hemoglobin concentration of 40, 80, 100, or 200 μg / dL (( 40, 80, 100 or 200 mg / dL expression level) / (0 mg / dL expression level)).

Next, of the miRNAs whose expression was detected, miRNAs that were stably detected in the high expression region were narrowed down.

Furthermore, miRNAs whose expression levels varied greatly depending on the degree of hemolysis were selected from the narrowed miRNAs. That is, there is a correlation between the increase in hemoglobin concentration and expression level (expression ratio), and the ratio between the expression level of hemoglobin concentration 0 mg / dL and the expression level of 40 mg / dL ((expression of hemoglobin concentration 40 mg / dL) MiRNA (SEQ ID NOs: 1 to 11) having an amount (quantity) / (0 mg / dL expression level)) of 2 or more was selected as a hemolysis marker. Table 1 shows the expression levels obtained from 11 selected miRNAs and serum samples with hemoglobin concentrations of 40, 80, 100, and 200 mg / dL.

The miRNAs (SEQ ID NOs: 1 to 11) shown in Table 1 increased in expression level as the hemoglobin concentration, that is, the degree of hemolysis increased, and the degree thereof was large. That is, it was found that miRNA shown in Table 1 can be used as a hemolysis marker, and hemolysis in a serum sample can be detected by measuring its expression level (abundance). In particular, hsa-miR-486-5p (SEQ ID NO: 6) and hsa-miR-92a-3p (SEQ ID NO: 10) show sensitive fluctuations even in slight hemolyzed samples with a hemoglobin concentration of 40 mg / dL. It was found that hemolysis can be detected with higher sensitivity.

(Threshold setting)
Blood was collected from 300 humans including normal and diseased, and serum was prepared. In the same manner as described above, RNA contained in each serum sample was extracted to obtain the abundance of various miRNAs. The abundance of various miRNAs was corrected using hsa-miR-149-3p (SEQ ID NO: 12), which is an endogenous miRNA, to obtain expression levels. For each hemolysis marker shown in Table 1, the median expression level of 2 samples + 2SD was determined, and this value was used as the threshold value 1 for the expression level of each hemolysis marker. Table 1 shows that when the expression level of each miRNA exceeds the threshold 1, the blood sample can be determined to be a hemolyzed sample.

As shown in Table 1, it was shown that all miRNAs represented by SEQ ID NOS: 1 to 11 of the present invention can detect hemolysis in a serum specimen having a hemoglobin concentration of 100 mg / dL or more. In particular, miRNAs represented by SEQ ID NOs: 1 to 5, 8, 9, and 11 are in serum samples having a hemoglobin concentration of 80 mg / dL or more, and miRNAs represented by SEQ ID NOs: 6 and 10 have a hemoglobin concentration of 40 mg / dL or more. It was shown that hemolysis can be detected with high sensitivity in each serum sample.

<Comparative Example 1>
In order to compare the hemolysis evaluation method of the blood sample of the present invention with the conventional method by visual observation, serum samples having hemoglobin concentrations of 0, 40, 80, 100, and 200 mg / dL used in Example 1 above are used. The presence or absence of hemolysis was evaluated by a method by visual observation.

As shown in Table 2, the serum specimen having a hemoglobin concentration of 200 mg / dL was observed to be reddish with the naked eye, and could be determined to be a hemolyzed specimen. On the other hand, in serum samples with hemoglobin concentrations of 40, 80, and 100 mg / dL, red color was not observable with the naked eye, and all were observed in almost the same color tone and could not be determined as hemolyzed samples. From this, it was found that hemolysis having a hemoglobin concentration of 100 mg / dL or less could not be detected by the conventional method by visual observation.

<Example 2>
Serum was prepared from blood that was aspirated into a vacuum blood collection tube vigorously from one healthy human, different from Example 1. As a result of measuring this serum sample with Eight Check-3WP (Sysmex Corporation), the hemoglobin concentration was 150 mg / dL.

Other than this, RNA was extracted in the same manner as in Example 1 to obtain the abundance of various miRNAs. This abundance was corrected using the abundance of endogenous miRNA hsa-miR-149-3p (SEQ ID NO: 12) to obtain the expression level of miRNA derived from serum samples. As a hemolysis marker, hsa-miR-486-5p (SEQ ID NO: 6) was selected, and when the expression level exceeded the threshold value 1 (275) obtained in Example 1, it was determined that the sample was a hemolysis sample. .

As a result, as shown in Table 3, the expression level of hsa-miR-486-5p derived from a serum sample was 1450, which exceeded the threshold value 1 of the hemolysis marker. Therefore, this serum sample was evaluated as a hemolysis sample. did.

From the above, it was confirmed that the hemolysis marker of the present invention, hsa-miR-486-5p, can correctly detect hemolysis in a serum sample having a hemoglobin concentration of 150 mg / dL.

<Example 3>
In the same manner as in Example 3, a serum sample of one healthy human was prepared. As a result of measuring this serum sample with Eight Check-3WP (Sysmex Corporation), the hemoglobin concentration was 100 mg / dL. Thereafter, in the same manner as in Example 2, the expression level of miRNA derived from a serum sample was obtained. Hsa-miR-486-5p (SEQ ID NO: 6) was selected as a hemolysis marker, and hemolysis was evaluated using threshold value 1.

As a result, as shown in Table 3, the expression level of hsa-miR-486-5p derived from the serum sample was 955, which exceeded the threshold value 275 of the hemolysis marker. Evaluated that there was.

From the above, it was confirmed that the hemolysis marker of the present invention, hsa-miR-486-5p, can correctly detect hemolysis of a serum sample having a hemoglobin concentration of 100 mg / dL. In general, even hemolysis with a hemoglobin concentration of about 100 mg / dL is said to affect the results of gene expression analysis (particularly in the case of miRNA expression analysis in blood samples). According to the marker of the present invention, hemolysis can be reliably detected even in a specimen having a hemoglobin concentration of 100 mg / dL, and it has been found that this is an effective method for obtaining an accurate gene expression analysis result.

<Example 4>
Serum specimens were prepared from blood that was slowly aspirated into a vacuum blood collection tube from one healthy human. As a result of measuring this serum sample with Eight Check-3WP (Sysmex Corporation), the hemoglobin concentration was 0 mg / dL. Except for this, the expression level of miRNA derived from a serum sample was obtained in the same manner as in Example 2. Hsa-miR-486-5p (SEQ ID NO: 6) was selected as a hemolysis marker, and hemolysis was evaluated using threshold value 1.

As a result, as shown in Table 3, the expression level of hsa-miR-486-5p derived from the serum sample was 120, which was below the threshold value 1 of 275 for the hemolysis marker, so no hemolysis was detected.

From the above, it was confirmed that hsa-miR-486-5p, which is a hemolysis marker of the present invention, can correctly detect a serum sample having a hemoglobin concentration of 0 mg / dL if it is not a hemolyzed sample.

<Comparative example 2>
Using the same serum sample having a hemoglobin concentration of 100 mg / dL as used in Example 3, the expression level of miRNA derived from the sample was obtained. Using hsa-miR-451a (SEQ ID NO: 29) described in Non-Patent Document 1 as a hemolysis marker, hemolysis was evaluated using threshold 1 as in Example 2. When the threshold value 1 of hsa-miR-451a was calculated as described in Example 1, it was 1774.

As a result, as shown in Table 4, the expression level of hsa-miR-451a was 1212, which was below the numerical value 1774 of the threshold value 1 of the miRNA, so that it could not be detected as a hemolyzed sample.

Therefore, the method for detecting hemolysis using hsa-miR-451a cannot detect hemolysis in a serum sample with a hemoglobin concentration of 100 mg / dL, which is thought to affect the results of gene expression analysis. It was confirmed that this is not a suitable method for obtaining results.

<Comparative Example 3>
Using the serum sample having a hemoglobin concentration of 200 mg / dL used in Example 1, the expression level of miRNA derived from the sample was obtained. Using hsa-miR-451a (SEQ ID NO: 29) described in Non-Patent Document 1 as a hemolysis marker, hemolysis was evaluated using threshold 1 as in Example 2.

As a result, as shown in Table 4, the expression level of hsa-miR-451a was 1182, which was lower than the numerical value 1774 of threshold value 1 of the miRNA, so that it could not be detected as a hemolyzed sample.

Therefore, the method for detecting hemolysis using hsa-miR-451a cannot detect hemolysis in a serum sample with a hemoglobin concentration of 200 mg / dL, which is thought to affect the results of gene expression analysis. It was confirmed that this is not a suitable method for obtaining results.

<Example 5>
Using the same serum sample having a hemoglobin concentration of 150 mg / dL as used in Example 2, the expression level of miRNA derived from the sample was obtained. Hsa-miR-642b-3p (SEQ ID NO: 9) was selected as a hemolysis marker, and its expression level was obtained in the same manner as in Example 2.

Here, a value that is 1.1 times the expression level obtained from the serum sample having a hemoglobin concentration of 80 mg / dL used in Example 1 is obtained, and this is used as the threshold value 2 for the expression level of each hemolytic marker. It was decided to evaluate that it was a hemolyzed specimen.

As a result, as shown in Table 5, the expression level of hsa-miR-642b-3p derived from the serum sample was 2686, which exceeded the threshold 2 of 1711 of the hemolysis marker. Evaluated that there was.

From the above, it was confirmed that hsa-miR-642b-3p, which is a hemolysis marker of the present invention, can correctly detect hemolysis in a serum sample having a hemoglobin concentration of 150 mg / dL.

<Example 6>
Using the same serum sample having a hemoglobin concentration of 100 mg / dL as used in Example 3, the expression level of miRNA derived from the sample was obtained. In the same manner as in Example 5, hsa-miR-642b-3p (SEQ ID NO: 9) was selected as a hemolysis marker, and hemolysis was evaluated using threshold value 2.

As a result, as shown in Table 5, the expression level of hsa-miR-642b-3p derived from the serum sample was 1782, which exceeded the threshold 2 of 1711 for the hemolysis marker. Evaluated that there was.

From the above, it was confirmed that the hemolysis marker of the present invention, hsa-miR-642b-3p, can correctly detect hemolysis of a serum sample having a hemoglobin concentration of 100 mg / dL. As described above, it is generally said that even hemolysis with a hemoglobin concentration of about 100 mg / dL has an effect on the results of gene expression analysis (particularly in the case of miRNA expression analysis in blood samples). According to the marker of the present invention, hemolysis can be reliably detected even in a specimen having a hemoglobin concentration of 100 mg / dL, and it has been found that this is an effective method for obtaining an accurate gene expression analysis result.

<Example 7>
Using the same serum sample having a hemoglobin concentration of 0 mg / dL as used in Example 4, the expression level of miRNA derived from the sample was obtained. In the same manner as in Example 5, hsa-miR-642b-3p (SEQ ID NO: 9) was selected as a hemolysis marker, and hemolysis was evaluated using threshold value 2.

As a result, as shown in Table 5, the expression level of the serum sample-derived hsa-miR-642b-3p was 512, which was lower than 1711 which is the threshold value 2 of the hemolysis marker, so hemolysis was not detected.

From the above, it was confirmed that hsa-miR-642b-3p, which is a hemolysis marker of the present invention, can correctly detect a serum sample having a hemoglobin concentration of 0 mg / dL if it is not a hemolyzed sample.

<Comparative example 4>
Using miRNA derived from a serum sample having the same hemoglobin concentration of 100 mg / dL as used in Example 3, and using hsa-miR-451a (SEQ ID NO: 29) described in Non-Patent Document 1 as a hemolysis marker As in Example 5, hemolysis was evaluated using threshold value 2. When the threshold value 2 of hsa-miR-451a was determined as described in Example 5, it was 1480.

As a result, as shown in Table 6, the expression level of hsa-miR-451a was 1200, which was below the value 1480 of threshold value 2 of the miRNA, so that it could not be detected as a hemolyzed sample.

Based on the above, the method for detecting hemolysis using hsa-miR-451a cannot detect hemolysis in a serum sample with a hemoglobin concentration of 100 mg / dL, which is thought to affect the results of gene expression analysis. It was confirmed that this is not a suitable method for obtaining the above.

Claims

MiR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073, miR-6131, miR-642b-3p, in blood samples A method for detecting hemolysis, comprising a measurement step of measuring the abundance of at least one miRNA selected from the group consisting of miR-92a-3p and miR-92b-3p.
miR-191-5p is hsa-miR-191-5p, miR-22-3p is hsa-miR-22-3p, miR-3158-5p is hsa-miR-3158-5p, miR- 320a is hsa-miR-320a, miR-484 is hsa-miR-484, miR-486-5p is hsa-miR-486-5p, miR-6073 is hsa-miR-6073, miR-6131 is hsa-miR-6131, miR-642b-3p is hsa-miR-642b-3p, miR-92a-3p is hsa-miR-92a-3p, and miR-92b-3p is The method of claim 1, which is hsa-miR-92b-3p.
The method for detecting hemolysis according to claim 1 or 2, wherein the miRNA is a polynucleotide shown in any of the following (a) to (e).
(a) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1 to 11 or a base sequence in which u is t in the base sequence;
(b) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1 to 11 or a base sequence in which u is t in the base sequence.
(c) a polynucleotide comprising a base sequence represented by any one of SEQ ID NOs: 1 to 11 or a base sequence complementary to a base sequence in which u is t in the base sequence.
(d) a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1 to 11 or a base sequence complementary to the base sequence in which u is t in the base sequence;
(e) a polynucleotide that hybridizes with the polynucleotide of any one of (a) to (d) under stringent conditions.
The method according to any one of claims 1 to 3, further comprising a correction step of correcting a measurement value of the abundance of the miRNA contained in a blood sample and obtaining a corrected measurement value as the expression level of the miRNA. .
The abundance or expression level of the miRNA in the blood sample, the difference between the abundance or expression level and the standard endogenous miRNA or external standard nucleic acid in the blood sample, or the abundance or expression level and the standard endogenous an evaluation step for evaluating that the blood sample is a hemolyzed sample when the ratio of the miRNA or the external standard nucleic acid in the blood sample exceeds a predetermined threshold as a reference for evaluating the presence or absence of hemolysis; The method according to any one of claims 1 to 4, comprising:
In the measurement step, a probe for capturing at least one miRNA selected from the miRNA immobilized on a support is contacted with a nucleic acid sample extracted from a blood sample and labeled with a labeling substance. The method according to any one of claims 1 to 5, which is a step of performing hybridization and measuring the abundance of the miRNA contained in a blood sample.
The measuring step includes measuring the abundance of a desired target miRNA contained in the blood sample at the same time as measuring the abundance of at least one miRNA selected from the miRNA contained in the blood sample. The method according to any one of 1 to 6.
MiR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073, miR- miRNAs for detecting hemolysis in blood samples A hemolysis detection chip comprising a support on which a probe for capturing at least one miRNA selected from the group consisting of 6131, miR-642b-3p, miR-92a-3p and miR-92b-3p is immobilized.
MiR-191-5p, miR-22-3p, miR-3158-5p, miR-320a, miR-484, miR-486-5p, miR-6073, miR- miRNAs for detecting hemolysis in blood samples A probe for capturing at least one miRNA selected from the group consisting of 6131, miR-642b-3p, miR-92a-3p and miR-92b-3p and a probe for capturing a desired target miRNA are immobilized. A chip for miRNA expression analysis including a support.