JP2021153487A - METHOD FOR PREPARING SERUM SAMPLE INCLUDING miRNA FROM BLOOD SAMPLE - Google Patents

Abstract

To provide a method for preparing a sample including miRNA, which allows precise measurement with small variation in measurement results, in order to accurately measure the abundance of the miRNA included in the blood sample.SOLUTION: Of the supernatant obtained by centrifugal separation of a blood sample, a middle layer is used to prepare a serum sample including miRNA, resulting in a serum sample allowing precise miRNA measurement.SELECTED DRAWING: Figure 2

Description

The present invention relates to a method for preparing a serum sample containing miRNA from a blood sample.

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

In recent years, miRNA is abundant not only in cells but also in body fluids such as whole blood, serum, plasma, urine, and cerebrospinal fluid, and its expression level can be a biomarker for various diseases including cancer. The sex is shown. As of December 2019, there are more than 2,600 types of miRNA in humans (miRBase release 22), and the expression of miRNA in body fluids can be expressed by using a measurement system such as a DNA microarray or next-generation sequencer (NGS). It can be detected and measured. Therefore, biomarker search studies targeting blood such as whole blood, serum, and plasma have been carried out using DNA microarrays or NGS, etc., and a genetic test using miRNA as a biomarker that can detect diseases at an early stage has been carried out. Expected to develop.

Since the measurement result of a genetic test greatly depends on the quality of the sample, the quality of the sample is extremely important for guaranteeing the measurement accuracy. However, the handling of specimens has not been sufficiently investigated in genetic testing. Among the genes, miRNA is particularly difficult to accurately quantify due to its low abundance in blood and short base pairs, and even if the same miRNA is measured with different blood sample quality, it is measured by the quantitative PCR method. Differences of 16 times or more may occur in the values (Non-Patent Document 1).

So Umemura, Biological Sample Analysis, 2016, Vol. 39, No. 5, p. 311-320

As described above, in order to accurately measure the abundance of miRNA contained in a blood sample, it is extremely important to prepare high-quality miRNA from the blood sample so that the variation in the measurement results is small. For example, as described in Non-Patent Document 1, in order to avoid the release of miRNA into a blood sample from a living tissue such as a blood cell, the sample is left for 5 hours or more after the sample is collected or the living tissue such as a blood cell is destroyed. By avoiding such methods, the quality of the prepared miRNA can be maintained to some extent. However, it was difficult to accurately measure the amount of miRNA even if they were carried out.

An object of the present invention is to find a method for preparing a sample containing miRNA capable of highly accurate measurement with little variation in measurement results in order to accurately measure the abundance of miRNA contained in a blood sample.

The present invention is a method for preparing a serum sample containing miRNA, which solves the above-mentioned problems, and comprises the following aspects (1) to (6).
(1) A method for preparing a serum sample containing miRNA from a blood sample.
Centrifugation step in which a blood sample is centrifuged using a columnar container having a cylindrical body and a bottom, and separated into a supernatant and a precipitate;
The supernatant separated in the centrifugation step is divided into three layers, an upper layer, a middle layer, and a lower layer, using the length in the height direction of the main body of the columnar container as an index. To obtain a serum sample containing miRNA, a middle layer separation step;
Preparation method including.
(2) The method according to (1), wherein the blood sample is whole blood, serum or plasma.
(3) The method according to (1) or (2), wherein the columnar container is a blood collection tube.
(4) The method according to any one of (1) to (3), wherein in the centrifugation step, a separating agent is contained in a columnar container together with a blood sample.
(5) The method according to (1) to (4), wherein the maximum centrifugal acceleration in the centrifugation step is 1200 xg or more and 3000 xg or less.
(6) A method for detecting a target miRNA contained in a blood sample.
Centrifugation step in which a blood sample is centrifuged using a columnar container having a cylindrical body and a bottom, and separated into a supernatant and a precipitate;
A classification step in which the supernatant separated in the centrifugation step is divided into three layers, an upper layer, a middle layer, and a lower layer, using the length in the height direction of the main body of the columnar container as an index.
The separated middle layer is separated to obtain a serum sample containing miRNA, a middle layer separation step; and the target miRNA contained in the serum sample obtained in the separation step is hybridized with a probe that specifically binds to the miRNA. Detection, detection process;
Detection methods, including.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to prepare a serum sample containing high-quality miRNA capable of highly accurate measurement of miRNA with small variation between measurements from a blood sample. Further, according to the present invention, the abundance of miRNA contained in a blood sample can be quantified more accurately than before. Therefore, for example, a more accurate disease test can be performed using the expression level of a biomarker in a blood sample as an index. It will be possible.

It is a figure which shows the example of the columnar container used in this invention. It is a figure explaining the step of dividing a supernatant after centrifugation of a blood sample into three layers, an upper layer, a middle layer and a lower layer. It is a graph which shows the average value of the coefficient of variation (CV value) obtained from the measurement signal of all miRNA contained in each serum sample of the upper layer, the middle layer and the lower layer prepared in Example 1. FIG.

The present invention is a method for preparing miRNA from a blood sample, which is a step of centrifuging a blood sample using a "cylindrical container" to separate it into a supernatant and a precipitate (centrifugation step), a "cylindrical container". Using the length of the main body in the height direction as an index, a step of dividing into three layers, an upper layer, a middle layer, and a lower layer (classification step), and a step of separating the divided middle layer to obtain a serum sample containing miRNA (middle layer separation). Step), a method including.

The method of the present invention prepares miRNA contained in a blood sample with high quality in gene expression analysis, for example, analysis using an array chip such as a microarray, or analysis by a polymerase chain reaction (PCR) method or a sequence method. , Can be used to perform these analyzes accurately. For gene expression analysis, for example, each miRNA is labeled with a miRNA in the blood and a support for capturing one or more target miRNAs and a probe for capturing the reference miRNA are fixed. To measure the expression level of the target miRNA by performing an amplification reaction using a probe for amplifying one or more target miRNAs and a probe for amplifying a reference miRNA, etc. In addition, these results are used to analyze and test gene expression, for example, to measure clinical specimens in order to understand the pathological condition.

"MiRNA" is a kind of non-coding RNA (ncRNA) which means a short RNA having a chain length of about 15 to 25 bases produced in a living body, and is considered to have a function of regulating the expression of mRNA. MiRNAs are transcribed from genomic DNA as RNAs (pre-mRNA) with a hairpin-like structure. This precursor is cleaved by a dsRNA cleaving enzyme (Drosha, Dicer) having a specific enzyme RNase III cleaving activity, then changes to a double-stranded form, and then becomes single-stranded. Then, one of the antisense strands is incorporated into a protein complex called RISC and is thought to be involved in the suppression of mRNA translation. As described above, since the mode of miRNA is different at each stage after transcription, usually, when miRNA is targeted (detection target), a hairpin structure, a double-stranded structure, a single-stranded structure, or the like is used. It is necessary to consider various forms. The existence of miRNA has been confirmed in various organisms.

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

The precipitate obtained when the blood sample is centrifuged is, for example, a blood clot if the blood sample is whole blood, and platelets or leukocytes remaining in a small amount in the serum or plasma if the blood sample is serum or plasma. ..

<Centrifugal separation process>
The centrifugation step of the present invention is a step of centrifuging a blood sample using a "cylindrical container" and separating it into a supernatant and a precipitate. Hereinafter, the columnar container used in the present invention will be described by way of illustration in FIGS. 1 (A) to 1 (D).

The columnar container 10 used in the present invention is a container having a main body portion 11 and a bottom portion 12 having a cylindrical shape. As shown in FIGS. 1A and 1C, the main body 11 has a circular shape or a substantially circular shape on the upper surface and the lower surface thereof, and has a columnar shape in which the diameters of the circles on the upper surface and the bottom surface are the same. As shown in 1 (B) and (D), the circles on the upper surface and the bottom surface may have different diameters in a truncated cone shape (in FIGS. 1 (B) and 1 (D), the diameter of the circles on the upper surface is the bottom surface. Larger than the diameter of the circle.). The bottom portion 12 is hemispherical or substantially hemispherical as shown in FIGS. 1 (A) and 1 (B), and has a conical pointed shape as shown in FIGS. 1 (C) and 1 (D). You may. In addition, the upper part of the container has an opening 13 for inflowing or outflowing a body fluid sample, and the opening can usually be closed by covering the inside of the container.

As the columnar container, for example, a blood collection tube, a centrifuge tube, or a microcentrifuge tube can be used. Of these, the blood collection tube is a columnar container for blood collection that is used in syringe blood collection and whose opening is pre-sealed with a lid so that the inside becomes negative pressure.

In the method of the present invention, among these columnar containers, the blood collection tube can be most preferably used. Specifically, as the blood collection tube, among Terumo's "Benoject II", a product with a blood collection volume of 10 ml and a diameter of 13.2 mm, a product with the same blood collection volume of 7 ml and a diameter of 13.2 mm, and a product with the same blood collection volume of 6 ml. 15.6 mm diameter product, 7.3 mm diameter product for 1 ml of blood collection; 16 mm diameter product for 10 ml blood collection volume, 16 mm diameter product for 7 ml blood collection volume of Becton Dickinson's "Vacutainer"; Of Sekisui Medical's "Insepack II", commercially available products such as a product with a blood collection volume of 10 ml and a diameter of 15.8 mm and a product with the same blood collection volume of 5 ml and a diameter of 12.7 mm can be used as they are.

In the centrifugation step, the columnar container can be used together with the blood sample containing a separating agent (also referred to as a serum separating agent) for preventing the precipitate from being mixed in the supernatant again after centrifugation. .. The separating agent is usually a gel-like substance, but changes to a highly fluid liquid during centrifugation. Therefore, after the completion of centrifugation, a septum is formed again between the supernatant of serum and the precipitate such as blood clot, and the separated supernatant is prevented from being mixed with the blood clot. And facilitates separation of the supernatant.

As the separating agent, polyester gel, polyolephen gel and the like are used. The separating agent may be one that is contained in the columnar container in advance, or may be added to the columnar container that does not contain the separating agent. The timing of adding the separating agent may be before centrifugation, before adding the blood sample to the columnar container, or after adding the blood sample.

The maximum centrifugal acceleration at the time of centrifugation is preferably 1200 xg or more and 3000 xg or less, and more preferably 2000 xg or more and 2600 xg or less.

The rotor of the centrifuge for centrifugation may be either a swing type or an angle type, but a swing type is preferable.

The temperature at the time of centrifugation may be any value, but the set temperature inside the centrifuge is preferably 4 ° C. or higher and 30 ° C. or lower, and more preferably 22 ° C. or higher and 26 ° C. or lower.

<Classification process>
The classification step of the present invention is a step of classifying the supernatant after centrifuging the blood sample into three layers, an upper layer, a middle layer, and a lower layer, using the length in the height direction of the main body of the columnar container as an index. be.

The classification of the centrifuged supernatant will be described by way of illustration in FIG.

The centrifuged supernatant is usually present in the main body of the columnar container. The supernatant can be classified using the length in the height direction of the container in which the supernatant is present as an index. Specifically, the length from the liquid level of the supernatant 1 to the boundary surface 6 with the precipitate 2 is measured using a scale displayed in advance on a ruler or a columnar container, and the upper layer 3 is measured according to a predetermined standard. , Middle layer 4 and lower layer 5 (FIG. 2 (A)). When the separating agent 7 is used at the time of centrifugation, the boundary surface 8 between the supernatant 1 and the separating agent 7 is used instead of the boundary surface 6 (FIG. 2 (B)). Here, the length from the liquid surface of the supernatant 1 to the boundary surface 6 with the precipitate 2 or the boundary surface 8 with the separating agent 7 is above the range because the main body of the columnar container has a cylindrical shape. It is a numerical value indicating the volume of Qing. Further, the length can be converted into a numerical value indicating the volume of the supernatant even when the main body of the columnar container has a truncated cone shape.

The criteria for classifying the upper layer, middle layer, and lower layer using the length in the height direction of the container in which the above supernatant is present as an index can be arbitrarily determined. For example, the criteria for classification when a general blood collection tube is used can be as follows. Alternatively, for convenience, the length of the supernatant portion may be divided into three equal parts and divided into an upper layer, a middle layer and a lower layer.

When the length of the supernatant portion is longer than 30 mm, the upper layer is preferably 3 mm to 15 mm from the liquid surface, more preferably 10 mm to 15 mm, and the lower layer is preferably 3 mm to 15 mm from the boundary surface, more preferably 10 mm. The thickness is from to 15 mm, and the middle layer can be divided as a layer sandwiched between the upper layer and the lower layer.

When the length of the supernatant portion is longer than 20 mm and 30 mm or less, the upper layer is preferably 1 mm to 10 mm from the liquid surface, more preferably 6 mm to 10 mm, and the lower layer is preferably 1 mm to 10 mm from the boundary surface. , More preferably from 6 mm to 10 mm, and the middle layer can be divided as a layer sandwiched between the upper layer and the lower layer.

When the length of the supernatant portion is longer than 10 mm and 20 mm or less, the upper layer is preferably from 0.6 mm to 7 mm, more preferably 3 mm to 7 mm from the liquid surface, and the lower layer is preferably from the boundary surface. It is set to 0.6 mm to 7 mm, more preferably 3 mm to 7 mm, and the middle layer can be classified as a layer sandwiched between the upper layer and the lower layer.

When the length of the supernatant portion is 10 mm or less, the upper layer is preferably 0.2 mm to 5 mm from the liquid surface, more preferably 1 mm to 5 mm, and the lower layer is 0.2 mm to 5 mm from the boundary surface. , More preferably from 1 mm to 5 mm, and the middle layer can be classified as a layer sandwiched between the upper layer and the lower layer.

More specifically, when using a product of Terumo's "Benoject II", which has a blood collection volume of 10 ml and a diameter of 13.2 mm, the upper layer is 10 mm from the liquid surface, the lower layer is 10 mm from the boundary surface, and the upper layer. The middle layer can be divided as a layer sandwiched between the lower layer and the lower layer.

In addition, Terumo's "Benoject II", which has a blood collection volume of 7 ml, has a diameter of 13.2 mm, Becton Dickinson's "Vacutainer", which has a blood collection volume of 10 ml and a diameter of 16 mm, or Sekisui Medical. When using a product with a diameter of 15.8 mm for a blood collection volume of 10 ml from the company's blood collection tube "Insepack II", the upper layer is 7 mm from the liquid surface and the lower layer is 7 mm from the boundary surface, as a layer sandwiched between the upper layer and the lower layer. The middle layer can be divided.

In addition, a product with a diameter of 15.6 mm for a blood collection volume of 6 ml from Terumo's blood collection tube "Benoject II" and a product with a diameter of 16 mm for a blood collection volume of 7 ml from Becton Dickinson's "Vacutainer" Or, when using "Insepack II", which is a blood collection tube of Sekisui Medical Co., Ltd., for a blood collection volume of 5 ml and a diameter of 12.7 mm, the upper layer is 5 mm from the liquid surface and the lower layer is 5 mm from the boundary surface, and is sandwiched between the upper layer and the lower layer. The middle layer can be divided as a layer.

In addition, when using a product with a diameter of 7.3 mm for 1 ml of blood collection volume from Terumo's blood collection tube "Benoject II", the upper layer is 1 mm in the longitudinal direction from the liquid surface and the lower layer is 1 mm in the longitudinal direction from the boundary surface. , The middle layer can be divided as a layer sandwiched between the upper layer and the lower layer.

<Middle layer separation process>
The middle layer separation step is a step of separating the middle layer classified in the classification step to obtain a serum sample containing miRNA.

The middle layer may be taken out from the columnar container after the separated upper layer is taken out, or only the middle layer may be taken out with the separated upper layer as it is.

To remove the upper layer from the cylindrical container, place the tip of the suction nozzle in the upper layer just below the liquid level, and suck the entire upper layer while lowering the position of the tip of the suction nozzle in one or more steps. It can be carried out.

After removing the upper layer, the tip of the suction nozzle is placed just below the liquid level of the middle layer, and the middle layer is sucked while lowering the position of the tip of the suction nozzle in one or more times. It can be done by discharging to the container of.

When taking out only the middle layer while leaving the separated upper layer as it is, place the tip of the suction nozzle near the center of the middle layer, and suck the middle layer while lowering the position of the tip of the suction nozzle in one or more times, and then use another. It can be done by discharging it into a container. In this case, the total amount of the middle layer to be sucked is preferably half or less, more preferably one-fourth or less of the amount of the middle layer in the supernatant.

The nozzle for sucking the supernatant may be made of metal or plastic.

The suction of the supernatant may be performed manually using a pipette or a dropper, or may be performed using a machine such as an electric pipette or an automatic dispenser.

Recovery of miRNA from a serum sample (middle layer) containing miRNA obtained in the above middle layer separation step can be performed by a known method (for example, the method of Fabalolo et al. (Favalolo et. Al., Methods Enzymol. 65: 718 (1980)). )) Etc.) can be applied. In addition, various commercially available kits for recovering miRNA (for example, "miRNeasy" manufactured by Qiagen, "3D-Gene" RNA extension reagent sample" manufactured by Toray Industries, etc.) can be applied.

<Detection process>
Detection of miRNA contained in a serum sample containing miRNA prepared by the method of the present invention is performed using, for example, an array chip such as a microarray in which a probe that specifically binds to the target miRNA is immobilized on a support. It can be carried out by measurement by a hybridization assay. For the measurement, an array chip containing a support on which a "miRNA capture probe" for capturing one or more miRNAs is immobilized can be used.

The term "capture probe" or "probe for capture" means a substance capable of directly or indirectly, preferably directly and selectively binding to a miRNA to be captured, as a typical example. , Nucleic acids, proteins, sugars and other antigenic compounds. 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, in the case of a nucleic acid, the derivative is a derivative labeled with a fluorescent group or the like, a modified nucleotide (for example, an alkyl such as halogen or methyl, an alkoxy such as methoxy, a nucleotide containing a group such as thio or carboxymethyl, and a re-base. It means a chemically modified derivative such as a derivative containing (such as a nucleotide having undergone composition, saturation of a double bond, deaminoization, substitution of an oxygen molecule with a sulfur molecule, etc.).

The chain length of the nucleic acid probe is preferably longer 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 exert selective binding to the target miRNA. Such an oligonucleic acid probe having a short chain length can be easily prepared by a well-known chemical synthesis method or the like.

Stringency during hybridization is known to be a function of temperature, salt concentration, probe chain length, GC content of the probe's nucleotide sequence and the concentration of chaotropic agents in the hybridization buffer. Stringent conditions include, for example, Sambook, J. et al. et al. (1998) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York, and the like can be used. The stringent temperature condition is about 30 ° C. or higher. Other conditions include hybridization time, concentration of detergent (for example, SDS), presence / absence of carrier DNA, and the like, and various stringencies can be set by combining these conditions. One of ordinary skill in the art can appropriately determine the conditions for obtaining the function as a capture probe prepared for the detection of the desired sample RNA.

Sequence information of miRNA can be obtained from databases such as GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and the website of miRBase (http://www.mirbase.org/). can. MiRNA capture probes 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 by using a number of miRNA capture probes immobilized on a support that covers all of the known miRNAs whose sequences have been identified, or for testing purposes, etc. Depending on the situation, a desired number of miRNA capture probes immobilized on the support may be used.

As the support on which the capture probe is aligned and immobilized, the same support as that used in known microarrays, macroarrays and the like can be used, and for example, slide glass, membranes, beads and the like can be used. can. It is also possible to use a support having a shape having a plurality of convex portions on the surface, which is described in Japanese Patent No. 4244788 and the like. The material of the support is not particularly limited, and examples thereof include inorganic materials such as glass, ceramics, and silicon; polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethylmethacrylate, and silicone rubber.

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

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). .. Since an organic solvent is used in the DNA synthesis reaction in these methods, it is desirable that the support is made of a material resistant to the organic solvent. Further, in the method of Francesco et al., Since the DNA synthesis is controlled by irradiating light from the back surface of the support, the support is preferably made of a translucent material.

Examples of the latter method include the method of Hirota et al. (Patent No. 3922454) and the method using a spotter. Examples of the spot method include a pin method by mechanically contacting the tip of a pin with a solid phase, an inkjet method using the principle of an inkjet printer, and a capillary method using a capillary tube. After the 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 bond on 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 a silane coupling agent treatment having an amino group or the like.

For hybridization with each miRNA capture probe immobilized on the support, a nucleic acid sample labeled with a labeling substance (nucleic acid sample derived from the sample) is prepared from RNA (sample RNA) extracted from the sample, and this labeling is performed. This is done by contacting the nucleic acid sample with the probe. The "nucleic acid sample derived from a sample" includes RNA extracted from the sample, as well as cDNA and cRNA prepared from the RNA by a reverse transcription reaction. The nucleic acid sample derived from the labeled sample may be a sample RNA directly or indirectly labeled 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.

As a method of binding the labeling substance to the nucleic acid sample derived from the sample, a method of binding the labeling substance to the 3'end of the nucleic acid sample, a method of binding the labeling substance to the 5'end, and a nucleotide to which the labeling substance is bound are incorporated into the nucleic acid. I can give you a way to make it. An enzymatic reaction can be used in the method of binding the labeling substance to the 3'end and the method of binding the labeling substance to the 5'end. For the enzymatic reaction, T4 RNA ligase, Thermal Deoxytyl Transferase, Poly A polymerase and the like can be used. For any of the labeling methods, the methods described in "Shao-Yao Ying ed., MiRNA Experimental Protocol, Yodosha, 2008" can be referred to. In addition, various kits for directly or indirectly binding a labeling substance to the end of RNA are commercially available. For example, as a kit for directly or indirectly binding a labeling substance to the 3'end, "3D-Gene" miRNA labeling kit (manufactured by Toray Industries, Inc.), "miRCURY miRNA HyPower labeling kit" (Exicon), Examples thereof include "NCode miRNA Labeling system" (Life Technologies), "FlashTag Biotin RNA Labeling Kit" (Genithia), and the like.

In addition, as in the conventional method, cDNA or cRNA incorporating the labeling substance is prepared by synthesizing cDNA or cRNA from the sample RNA in the presence of labeled deoxyribonucleotide or labeled ribonucleotide, and the cDNA or cRNA incorporating the labeling substance is prepared and arrayed. It is also possible to hybridize with the above probe.

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

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

The nucleic acid sample derived from the sample labeled as described above is brought into contact with the miRNA capture probe on the support to hybridize the nucleic acid sample and the probe. This hybridization step can be performed in exactly the same manner as before. 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 to 70 ° C. for 1 minute to a dozen 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 by 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, a fluorescence scanner, or the like may be used.

The measured fluorescence intensity measured is compared to ambient noise. Specifically, the measured values obtained from the probe-immobilized region are compared with the measured values obtained from other positions, and the case where the former value is exceeded is regarded as detected (valid judgment positive). ..

If the detected measured value contains background noise, the background noise may be subtracted. Ambient noise can also be subtracted from the detected measurements as background noise. In addition, the method described in "Kou Fujibuchi, Katsuhisa Horimoto, Microarray Data Statistical Analysis Protocol, Yodosha, 2008" may be used.

The abundance of miRNA contained in the sample can be calculated by using the measured value obtained in the above measurement as it is. For example, when the gene expression analysis of miRNA contained in the serum sample is performed, it is described below. The corrected abundance amount may be calculated by correcting the measured value by various illustrated methods.

As the correction method, a conventional method can be used, and examples thereof include a global normalization method and a quantum normalization method in which correction is performed using the measured values of all the detected miRNAs. Further, correction may be performed using housekeeping RNA such as U1 snoRNA, U2 snoRNA, U3 snoRNA, U4 snoRNA, U5 snoRNA, U6 snoRNA, 5S rRNA, 5.8S rRNA, or a specific endogenous miRNA for correction. It may be corrected by using an external standard nucleic acid added at the time of RNA extraction or labeling. By "endogenous" is meant that it is not artificially added to the specimen, but is naturally present in the specimen. For example, the term "endogenous miRNA" refers to a miRNA that is naturally present in a sample and is derived from the organism that provided the sample. When the gene expression analysis of the target miRNA contained in the serum sample is performed by applying the method of the present invention, it is preferable to use a correction method using an external standard nucleic acid such as spike control that does not depend on the sample.

<Example 1>
Hereinafter, the process of preparing a serum sample containing miRNA by the method of the present invention, measuring the miRNA signal, and calculating the measurement accuracy will be described more specifically. However, the present invention is not limited to the following examples.

(DNA microarray)
The following experiments were carried out using "3D-Gene" human miRNA oligo chip (corresponding to miRBase release 21) manufactured by Toray Industries, Inc.

(Preparation of miRNA)
Blood was collected from healthy subjects using a blood collection tube with a diameter of 15.6 mm for a blood collection volume of 6 ml from Terumo's "Benoject II". After blood collection, the blood was allowed to stand at room temperature for 30 minutes or more, and the blood collection tube containing whole blood was centrifuged at 24 ° C. and 2300 xg for 10 minutes to separate into 2 ml of supernatant and precipitate. The blood collection tube was erected vertically, the upper layer was 5 mm in the longitudinal direction from the liquid surface of the supernatant, and the lower layer was 5 mm in the longitudinal direction from the interface with the precipitate, and the middle layer was divided as a layer sandwiched between the upper layer and the lower layer. The upper layer, middle layer and lower layer were sequentially transferred to separate containers and stored at −80 ° C. The upper layer, middle layer and lower layer of the stored supernatant were thawed, 300 μL of each sample was dispensed into three, and miRNA (hereinafter referred to as sample miRNA) was extracted from each. For the extraction, "3D-Gene" RNA extraction reagent sample kit "(Toray Industries, Inc.) was used.

(Detection of miRNA)
The obtained sample miRNA was labeled with a "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.) to measure the fluorescence intensity. The scanner was set to 100% laser output, and the voltage setting of the photo multiplier was set to AUTO.

Samples dispensed into three from the serum samples of the upper layer, middle layer, and lower layer were measured, and measurement signals of all miRNAs (79 types) detected by the DNA microarray were obtained. The standard deviation of all miRNAs in each layer was calculated from the miRNA signals of 3 samples in each layer. The calculated standard deviation of each miRNA was divided by the signal average value of each miRNA to calculate the CV value of each miRNA. The average of the CV values of all miRNAs in each layer was calculated, and the measurement accuracy of each layer was obtained. Using the CV values of all miRNAs in each layer, t-test was performed for the upper layer and the middle layer, and the lower layer and the middle layer.

As a result, as shown in FIG. 3, the CV value of the middle layer was significantly lower than the CV value of the miRNA of the upper layer and the lower layer. From this, among the supernatants obtained by centrifuging blood samples, by preparing serum samples containing miRNA using the middle layer, highly accurate miRNA with little variation in measured values between measurements can be obtained. It turns out that measurement is possible.

1 Supernatant 2 Precipitate 3 Upper layer 4 Middle layer 5 Lower layer 6 Interface between supernatant and precipitate 7 Separator 8 Interface between supernatant and separator 10 Cylindrical container 11 Main body 12 Bottom 13 Aperture

Claims (6)

A method for preparing a serum sample containing miRNA from a blood sample.
Centrifugation step in which a blood sample is centrifuged using a columnar container having a cylindrical body and a bottom, and separated into a supernatant and a precipitate;
The supernatant separated in the centrifugation step is divided into three layers, an upper layer, a middle layer, and a lower layer, using the length in the height direction of the main body of the columnar container as an index. To obtain a serum sample containing miRNA, a middle layer separation step;
Preparation method including. The method of claim 1, wherein the blood sample is whole blood, serum or plasma. The method according to claim 1 or 2, wherein the columnar container is a blood collection tube. The method according to any one of claims 1 to 3, wherein in the centrifugation step, the separating agent is contained in a columnar container together with the blood sample. The method according to any one of claims 1 to 4, wherein the maximum centrifugal acceleration at the time of centrifugation is 1200 xg or more and 3000 xg or less. A method for detecting target miRNA contained in blood samples.
Centrifugation step in which a blood sample is centrifuged using a columnar container having a cylindrical body and a bottom, and separated into a supernatant and a precipitate;
A classification step in which the supernatant separated in the centrifugation step is divided into three layers, an upper layer, a middle layer, and a lower layer, using the length in the height direction of the main body of the columnar container as an index.
A middle layer separation step of separating the separated middle layer to obtain a serum sample containing miRNA; and hybridization of the target miRNA contained in the serum sample obtained in the middle layer separation step with a probe that specifically binds to the miRNA. Detection process;
Detection methods, including.

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