JP2021119752A - Method for determining quality of hybridization reaction of nucleic acid - Google Patents

Abstract

To provide a method for determining the quality of hybridization reaction of a nucleic acid under detection by individual measurement data of a DNA chip.SOLUTION: Using an amount of hybridization of a specific micro RNA as an index, the quality of hybridization reaction of a nucleic acid under detection is evaluated.SELECTED DRAWING: None

Description

The present invention relates to a method for determining the quality of a hybridization reaction between a nucleic acid to be measured and a probe immobilized on a substrate in the detection of nucleic acid in a sample using a DNA chip.

Nucleic acids represented by DNA and RNA play a role as signal transmitters in the living body, and their detection is important in elucidation of biological functions and diagnosis of diseases.

A DNA chip is known as a tool for comprehensively detecting nucleic acids at one time. A plurality of probes that specifically capture the nucleic acid to be measured by hybridization are immobilized on the substrate of the DNA chip, and the hybridization reaction is carried out by contacting a solution containing the nucleic acid to be measured. Wake up and an immobilized probe captures and detects nucleic acids in solution. Therefore, the detection sensitivity of the DNA chip greatly depends on the quality of the hybridization reaction. That is, by improving the efficiency of the hybridization reaction, it is possible to improve the detection sensitivity of the DNA chip. For example, Patent Document 1 discloses a method of stirring a solution on a DNA chip by moving fine particles or bubbles as a method of improving the efficiency of the hybridization reaction, and has succeeded in improving the detection sensitivity.

International Publication No. 2005/090997

As described above, the detection sensitivity of the DNA chip largely depends on the efficiency of the hybridization reaction. On the other hand, the quality of the hybridization reaction is also affected by the reaction conditions such as the time and temperature at which the DNA chip and the solution are brought into contact with each other and the presence or absence of stirring. Therefore, if the reaction conditions change due to a malfunction of the apparatus or a mistake of the operator, the measurement data of the DNA chip is affected. Until now, there was no simple method for determining the quality of the hybridization reaction from the individual measurement data of the DNA chip, and the validity of the measurement data could not be determined.

An object of the present invention is to find a method for determining the quality or quality of a hybridization reaction from the measurement data of a DNA chip.

In order to solve the above problems, the present inventors use the hybridization amount of a specific microRNA (hereinafter, also referred to as miRNA) among nucleic acids as an index to determine the quality or quality of the hybridization reaction of the nucleic acid to be measured. The present invention was completed by finding that it can be evaluated. In the present invention, the miRNAs shown in SEQ ID NOs: 1 to 6 are used as "determination miRNAs" as miRNAs in which the efficiency of the hybridization reaction fluctuates relatively greatly under the influence of hybridization reaction conditions. This is a method for determining or evaluating the quality or quality of the hybridization reaction of the nucleic acid to be measured by comparing it with the hybridization amount of a predetermined reference miRNA (hereinafter referred to as reference miRNA). It consists of the following aspects.
(1) A method for determining the quality of the hybridization reaction of the nucleic acid to be detected in the detection of nucleic acid in a sample using a DNA chip.
DNA on which one or more determination miRNAs selected from miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6 and a probe that hybridizes with one or more predetermined reference miRNAs are immobilized on the substrate. A reaction step in which a sample is brought into contact with the chip to carry out a hybridization reaction;
A measurement step of measuring the hybridization amount of one or more determination miRNAs obtained in the reaction step and the hybridization amount of one or more reference miRNAs; and one or more determinations obtained in the measurement step. A determination step of comparing the hybridization amount of the target miRNA with the hybridization amount of one or more reference miRNAs to determine the quality of the hybridization reaction of the nucleic acid to be detected;
How to include.
(2) The method according to (1), wherein the sample is agitated using fine particles or bubbles in the reaction step.
(3) The method according to (2), wherein in the reaction step, the sample is agitated by moving the fine particles by gravity or centrifugal force.
(4) In the determination step, when the difference or ratio between the hybridization amount of one or more determination miRNAs and the hybridization amount of one or more reference miRNAs exceeds a predetermined threshold as a reference, the hybridization reaction occurs. The method according to any one of (1) to (3), which is determined to be good.
(5) The method according to any one of (1) to (4), wherein the nucleic acid to be detected is miRNA.
(6) The method according to any one of (1) to (5), wherein the sample is blood, serum or plasma.
(7) A DNA chip for quality determination of the hybridization reaction of the nucleic acid to be detected.
A probe for capturing a quality-determining miRNA for one or more hybridization reactions selected from miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6, and a probe for capturing one or more reference miRNAs. A DNA chip in which a probe is immobilized on a substrate.

According to the present invention, it is possible to determine the quality of the hybridization reaction from the measurement data of the DNA chip, which has been difficult until now. By using the present invention, it becomes possible to find the measurement data in which the hybridization reaction is poor for some reason from the individual measurement data of the DNA chip, and the reliability of the measurement data is improved.

The present invention is a method for determining the quality or quality of the hybridization reaction of the nucleic acid to be detected in the detection of nucleic acid in a sample using a DNA chip.
A DNA chip on which a probe that hybridizes with one or more determination miRNAs selected from miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6 and one or more predetermined reference miRNAs is immobilized on a substrate. A reaction step in which a sample is brought into contact with the sample to carry out a hybridization reaction;
A measurement step of measuring one or more determination miRNA hybridization amounts and one or more reference miRNA hybridization amounts obtained in the reaction step; and one or more determination amounts obtained in the measurement step. A determination step of comparing the amount of hybridization of miRNA with the amount of hybridization of one or more reference miRNAs to determine the quality of the hybridization reaction of the nucleic acid to be detected;
It is a method including.

Here, the "quality" of the nucleic acid hybridization reaction is synonymous with the "good or bad" of the nucleic acid hybridization reaction, and can be paraphrased with each other in the present specification.

The DNA chip of the present invention is a chip in which one or a plurality of probes that specifically capture the nucleic acid to be measured by hybridization are immobilized on a substrate that is a carrier (support), and the nucleic acid to be measured is the nucleic acid to be measured. The hybridization reaction is carried out by contacting the solution containing the nucleic acid, and the immobilized probe can capture and detect the nucleic acid to be measured in the solution. As the probe, in addition to DNA and RNA that hybridize with the nucleic acid to be measured, nucleic acid derivatives such as PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid) are used. From the viewpoint of hybridization stability, the base length of the probe is preferably 20 to 100. The probe preferably has a base sequence that is completely complementary to the nucleic acid to be measured, but even if there are some differences, it has a highly homologous base sequence and selectively binds to the nucleic acid to be measured. If possible, it can be used as a probe.

As the substrate (carrier on which the probe is immobilized) used for the DNA chip, the same substrate as that used in known microarrays, macroarrays and the like can be used, and for example, slide glass, membranes and beads can be used. Etc. can be used. It is also possible to use a substrate 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 substrate is not particularly limited, and examples thereof include inorganic materials such as glass, ceramic, and silicon; and resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and silicone rubber.

As a method of immobilizing the probe on the substrate, a method of synthesizing an oligo DNA on the surface of the substrate and a method of dropping and immobilizing the oligo DNA synthesized in advance on the surface of the substrate 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). .. In these methods, since an organic solvent is used during the DNA synthesis reaction, it is desirable that the substrate used as the support is 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 substrate, the substrate 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 substrate, 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-treated substrate, 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 substrate is usually performed by a silane coupling agent treatment having an amino group or the like.

The sample that can be used in the present invention is not particularly limited, and includes, for example, body fluids such as blood, serum, plasma, urine, spinal fluid, saliva, cerebrospinal fluid, sweat, tears, semen, lymph, and tissue fluid. , Frozen specimens of various tissues or cells, paraffin-embedded specimens (FFPE) or sections thereof, culture fluids in which cells or tissues are cultured, and the like, that is, specimens isolated from living organisms, that is, biological samples. Among these samples, in the present invention, body fluid samples such as blood, serum, plasma, urine, cerebrospinal fluid, saliva, swab or various tissue fluids are preferably used, and blood, serum or plasma is particularly preferable.

Examples of the treatment of the body fluid include extraction of the nucleic acid to be detected from the body fluid. Nucleic acid extraction is performed by a known method (for example, the method of Fabalolo et al. (Favalolo et. Al., Methods Enzymol. 65: 718 (1980)), etc.), and various kits for that purpose are also commercially available (for example, of Qiagen). miRNeasy, Toray Industries, Inc.'s "3D-Gene" RNA extraction reagent sample, etc.) can be applied.

Examples of the nucleic acid derived from the sample to be detected include cell-free DNA, genomic DNA, messenger RNA, microRNA (hereinafter, miRNA), cDNA prepared by reverse transcription reaction from these, cRNA, and the like, and miRNA is particularly used. preferable. MiRNA is a kind of non-coding RNA which means a short RNA having a strand length of about 15 to 25 bases produced in a living body, and is considered to have a function of regulating the expression of messenger RNA. Further, the nucleic acid to be detected may be one labeled in advance with a fluorescent substance or the like.

As a method of binding the labeling substance to the nucleic acid to be detected, 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. Methods and the like can be mentioned. 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 (Toray), miRCURY miRNA HyPower labeling kit (Exicon), NCode miRNA Labeling system (Life Technologies). , FlashTag Biotin RNA Labeling Kit (Jennsphere) and the like can be exemplified.

In addition, as in the conventional method, cDNA or cRNA incorporating a labeling substance is prepared by synthesizing cDNA or cRNA from RNA in the presence of labeled deoxyribonucleotide or labeled ribonucleotide, and this is placed on an array. It is also possible to hybridize with the probe of.

Examples of the labeling substance that can be used in the present invention 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. Fluorescent dyes that are easy to measure and easy to detect are preferred. Specifically, cyanine (cyanine 2), aminomethylcoumarin, fluorosane, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, 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).

In the present invention, the quality of the hybridization reaction of the nucleic acid to be detected is determined. The quality of the hybridization reaction is based on the quality of efficiency at which the probe is captured by hybridization with the nucleic acid to be detected. The quality of the hybridization reaction is affected by hybridization conditions such as reaction time, reaction temperature, and stirring efficiency of the solution during the reaction. In general, the efficiency of the hybridization reaction is higher when the reaction time is long, when the reaction temperature is low, and when the stirring efficiency is high.

The miRNA used to determine the quality of the hybridization reaction in the present invention, that is, the "determination miRNA" is selected as a miRNA whose effect on the efficiency of the hybridization reaction itself varies greatly depending on the hybridization reaction conditions. Will be done. Specifically, it is a miRNA composed of the nucleotide sequences shown in SEQ ID NOs: 1 to 6.

The miRNA used as a criterion for determining the quality of the hybridization reaction in the present invention, that is, the "reference miRNA" is a miRNA other than the determination miRNA, which is arbitrarily determined in advance. In general, when measuring the amount of hybridization of a target nucleic acid, it may be affected by conditions other than the hybridization reaction conditions such as the type of sample and the processing method. Therefore, the target nucleic acid is usually less affected by such conditions. Nucleic acid similar to the above is simultaneously hybridized, and the measured value of the target nucleic acid is corrected and used using the measured value as a reference value. Also in the present invention, in addition to measuring the hybridization amount of the determination miRNA, a miRNA contained in the same sample and having a relatively small effect on the efficiency of the hybridization reaction due to the hybridization reaction conditions is used as the reference miRNA. For the purpose, the reference miRNA is preferably a miRNA (non-degraded endogenous miRNA) that is stably present in the sample. Examples of non-degraded endogenous miRNAs include miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 7 to 9.

<Reaction process>
In the reaction step, a sample containing the nucleic acid to be detected is brought into contact with a DNA chip in which one or more probes that specifically capture the determination miRNA and the reference miRNA by hybridization are immobilized on the substrate, and hybridization is performed. This is a step of performing a reaction and causing the probe to capture the determination miRNA and the reference miRNA.

These determination miRNA and reference miRNA can be extracted from the sample by the above-mentioned method in the same manner as the nucleic acid to be detected. Further, like the nucleic acid to be detected, the nucleic acid may be pre-labeled with a fluorescent substance or the like by the above-mentioned method.

The sample containing the nucleic acid to be detected may contain a nucleic acid other than the nucleic acid to be detected.

The composition of the sample solution can affect the quality of the hybridization reaction. A general solution composition includes a buffer, a salt, and a surfactant.

The time (reaction time) and temperature (reaction temperature) at which the sample is brought into contact with the DNA chip can affect the quality of the hybridization reaction. The general reaction time is 1 minute to a dozen hours, and the general reaction temperature is about 20 ° C. to 70 ° C.

The sample may be simply brought into contact with the DNA chip while the DNA chip is left to stand, but it is preferable to stir the sample in order to increase the efficiency of the hybridization reaction. As a method of stirring the sample, for example, fine particles or bubbles are mixed with the sample and the DNA chips are moved to move them, or only the DNA chips are moved without using the fine particles or bubbles. The method can be mentioned.

The size of the fine particles (maximum diameter of the fine particles) used for stirring the sample is preferably 10 μm or more. If the size of the fine particles is smaller than 10 μm, the effect of stirring by the fine particles may be hardly obtained. The reason for this is that if the size of the fine particles is smaller than 10 μm, the fine particles may hardly move even if an external field (magnetic field, gravity, or vibration) is applied due to the resistance of the solution. The size of the fine particles is particularly preferably 20 μm or more.

The shape of the fine particles used for stirring the sample is preferably spherical, that is, beads. It is preferable that the fine particles are beads because they can roll smoothly and move smoothly in the reaction solution without stagnation, and as a result, the sample solution can be well stirred. Most preferably, spherical fine particles (microbeads) having a diameter of 20 μm to 300 μm can be used as the form of the fine particles. When the diameter of the beads is within this range, even if there is resistance of the solution due to the weight of the beads themselves, the beads can easily move in the liquid due to gravity or acceleration, and the solution can be sufficiently agitated, resulting in good results. Can be obtained.

As the material of the fine particles used for stirring the sample, metal, glass, ceramic, polymer (polystyrene, polypropylene, nylon, etc.) can be used. Among these, beads made of a material (glass, quartz, zirconia ceramic) having a specific gravity higher than that of water are preferable because they can easily move in the liquid due to acceleration due to gravity or vibration. It is also possible to use magnetic beads. In particular, beads made of zirconia ceramic can be most preferably used because they have a large specific gravity and can be easily moved by acceleration due to gravity or vibration. Further, glass, quartz, and zirconia ceramic are preferable because the bead component is less likely to be eluted in the sample solution. Beads made of zirconia ceramic (yttria stabilized zirconia) is the density is larger than that, such as 2.2 g / cm ³ of 6 g / cm @ 3 and the quartz glass, the stirring effect can be exhibited more, the solution at the time of sealing with the container Since the beads often fly up and move with respect to movement, setting can be performed more easily, which is particularly preferable.

In the method of moving the fine particles and stirring the sample, a method of moving the fine particles by any one of centrifugal force, gravity, magnetic force, vibration of the carrier, or a combination thereof is preferable.

Examples of the method of moving the fine particles by centrifugal force to agitate the sample include a method of swirling the DNA chip along a horizontal plane. The turning speed at this time is preferably 50 rpm to 1000 rpm. For example, a method of swirling the DNA chip in a horizontal plane using a bioshaker can be used.

Examples of the method of moving the fine particles by gravity to agitate the sample include a method of rotating the DNA chip along a vertical plane (a plane parallel to the direction of gravity). The rotation speed at this time is preferably 0.1 rpm to 30 rpm. For example, a method of swirling in a vertical plane using a microtube rotator can be used.

When moving only the DNA chip without using fine particles or bubbles, a method of rotating the DNA chip using a bioshaker, a microtube rotator, or the like can be similarly used.

<Measurement process>
In the measurement step, the hybridization amount of the determination miRNA obtained in the reaction step and the hybridization amount of the reference miRNA are measured. At the same time, the amount of hybridization of the nucleic acid to be detected may be measured.

In the measurement of the amount of hybridization, the signal value of the probe-immobilized region on the substrate is detected, and the amount of the determination miRNA and the reference miRNA captured by hybridization on the probe is measured using the detected signal intensity as an index. ..

The signal value is detected by using an appropriate signal reading device according to the type of the labeling substance labeled on the determination miRNA and the reference miRNA. As the labeling substance, various labeling substances such as fluorescent dyes, phosphorescent dyes, enzymes, and radioisotopes used in known microphone mouth array analysis can be used. When a fluorescent dye is used as a labeling substance, the fluorescence signal may be detected using a fluorescence microscope, a fluorescence scanner, or the like.

The detected signal value is compared with the signal value (noise value) other than the probe immobilization region on the substrate. If the signal value exceeds the noise value, that signal value is valid. If the detected signal value includes a noise value, the noise value may be subtracted.

<Judgment process>
In the determination step, the quality of the hybridization reaction is determined by comparing the hybridization amount of one or more determination miRNAs measured in the measurement step with the hybridization amount of one or more reference miRNAs.

As a specific example, the quality of the hybridization reaction can be determined by using the difference or ratio of the hybridization amounts of the determination miRNA and the reference miRNA. In this case, a threshold value as a reference for determination is set in advance, and the quality or quality (good or bad) of the hybridization reaction can be determined depending on whether or not the difference or ratio of the hybridization amount exceeds the threshold value. .. That is, when the difference or ratio of the hybridization amount between the determination miRNA and the reference miRNA exceeds the reference threshold arbitrarily determined in advance, it is determined that the hybridization reaction is good, and the determination miRNA and the reference miRNA are used for reference. When the difference or ratio of the hybridization amount of miRNA is equal to or less than the reference threshold, it can be determined that the hybridization reaction is poor.

In the determination step, the difference or ratio of the hybridization amount between the determination miRNA and the reference miRNA may be converted into a logarithm, and the determination may be performed using the logarithmic value.

As the determination miRNA, one or more miRNAs may be arbitrarily selected from the miRNAs shown in SEQ ID NOs: 1 to 6. For example, when using one determination miRNA, one of hsa-miR-4454 (SEQ ID NO: 1), hsa-miR-6131 (SEQ ID NO: 2), and hsa-miR-451a (SEQ ID NO: 3) is selected. It is preferable to do so. Further, when performing more rigorous or highly accurate evaluation, it is preferable to use a plurality of determination miRNAs. When a plurality of determination miRNAs are used, the hybridization amount of each miRNA may be used, or the average value or the median value of the hybridization amount may be used as a representative value.

As the reference miRNA, one or more miRNAs may be arbitrarily selected from the predetermined miRNAs. For example, it is preferable to select and use from non-degraded endogenous miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 7 to 9. When using one reference miRNA, select one of hsa-miR-149-3p (SEQ ID NO: 7), hsa-miR-2861 (SEQ ID NO: 8), and hsa-miR-4463 (SEQ ID NO: 9). It is preferable to do so. Further, when performing more rigorous or highly accurate evaluation, it is preferable to use a plurality of reference miRNAs. When a plurality of reference miRNAs are used, the hybridization amount of each miRNA may be used, or the average value or the median value of the hybridization amount may be used as a representative value.

The threshold value as a criterion for judgment can be arbitrarily set according to the purpose of evaluation, the required accuracy, and the like.

When one determination miRNA and one reference miRNA are used, for example, as shown in Formula 1A, the hybridization amount e of the determination miRNA and the hybridization amount E of the reference miRNA are measured, and the hybridization thereof is performed. The amount ratio (e / E) is determined, and when this value exceeds the threshold value t1, the hybridization reaction can be determined to be good.

e / E> t1 (Equation 1A).

Further, as shown in the formula 2A, the difference (e-E) in the measurement of the hybridization amount e of the determination miRNA and the hybridization amount E of the reference miRNA is obtained, and when this value exceeds the threshold value t2, the hybridization reaction is performed. Can be judged to be good.

e-E> t2 (Equation 2A).

When used as a plurality of determination miRNAs and a plurality of reference miRNAs, a representative value of the hybridization amount of the plurality of determination miRNAs and a representative value of the hybridization amount of the plurality of reference miRNAs are obtained, and both representative values are used. It can be used to find the difference or ratio. Specifically, in the determination criteria shown in the above formulas 1A and 2A, instead of the hybridization amount e of the determination miRNA, a representative value r of the hybridization amount of a plurality of determination miRNAs is used as the high of the reference miRNA. By using the representative value R of the hybridization amount of the plurality of reference miRNAs instead of the hybridization amount E, the determination can be made using the formula 1B or the formula 2B. As the representative value, the average value or the median value can be used.

r / R> t1 (Equation 1B)
r-R> t2 (Equation 2B).

When used as one determination miRNA and a plurality of reference miRNAs, the formula 1C or the formula 2C can be obtained by using the typical value R of the hybridization amount e of the determination miRNA and the hybridization amount of the plurality of reference miRNAs. Can be determined using.

e / R> t1 (Equation 1C)
e-R> t2 (Equation 2C).

When used as one determination miRNA and a plurality of reference miRNAs, the formula 1D or the formula 2D can be obtained by using the representative value r of the hybridization amount of the plurality of determination miRNAs and the hybridization amount E of the reference miRNAs. Can be determined using.

r / E> t1 (Equation 1D)
r-E> t2 (Equation 2D).

Here, in the above equations 1A, 2A, 1B, 2B, 1C, 2C, 1D and 2D, the width of a constant error α within the threshold values t1 and t2 in consideration of experimental errors and the like. May be provided as "t1 ± α" and "t2 ± α", respectively. The error α in this case may be set arbitrarily. For example, in the formula 2A, about 10% of E can be set as α to give a range to the threshold value t2.

Further, as the threshold value of each abundance amount, a value obtained by converting the value of the hybridization amount into a logarithm may be used. In this case, an appropriate threshold value may be set according to the conversion. For example, when the formula 1A is applied, the ratio (e / E) of the hybridization amount of the determination miRNA to the hybridization amount of the reference miRNA is converted into a logarithmic value, and the threshold value t1 is set according to the conversion. good.

In addition, the difference or ratio of the amount of hybridization with the reference miRNA is obtained for each determination miRNA, and the determination is made according to the determination criteria for each determination miRNA, and the results are integrated to determine the quality of the hybridization reaction. can do.

Specifically, for example, in the determination for each determination miRNA, when the number of determination miRNAs determined to be good exceeds the number of determination miRNAs determined to be poor or an arbitrary predetermined number, a hybridization reaction occurs. Can be judged to be good. On the contrary, when the number of determination miRNAs determined to be defective exceeds the number of determination miRNAs determined to be good or a predetermined number, the hybridization reaction can be determined to be defective.

In the present invention, the miRNA consisting of the nucleotide sequences represented by SEQ ID NOs: 1 to 6 described below is used as a determination miRNA for determining hybridization.

The term "hsa-miR-4454 gene" or "hsa-miR-4454" used as a determination miRNA herein refers to the hsa-miR-4454 gene (miRBase Accession) consisting of the nucleotide sequence set forth in SEQ ID NO: 1. No. MIMAT0018976) and other species such as homologs or orthologs are included. The hsa-miR-4454 gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-4454" includes a sequence of "hsa-mir-4454" (miRBase Accession No. MI0016800, SEQ ID NO: 10) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-6131 gene" or "hsa-miR-6131" used as a determination miRNA in the present specification refers to the hsa-miR-6131 gene (miRBase Accession) consisting of the nucleotide sequence shown in SEQ ID NO: 2. Includes No. MIMAT0024615) and other species homologs or orthologs. The hsa-miR-6131 gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-6131" includes a sequence of "hsa-mir-6131" (miRBase Accession No. MI0021276, SEQ ID NO: 11) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-451a gene" or "hsa-miR-451a" used as a determination miRNA in the present specification refers to the hsa-miR-451a gene (miRBase Accession) consisting of the nucleotide sequence shown in SEQ ID NO: 3. Includes No. MIMAT0001631) and other species homologs or orthologs. The hsa-miR-451a gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-451a" includes a sequence of "hsa-mir-451a" (miRBase Accession No. MI0001729, SEQ ID NO: 12) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-1260b gene" or "hsa-miR-1260b" used as a determination miRNA in the present specification refers to the hsa-miR-1260b gene (miRBase Accession) consisting of the nucleotide sequence shown in SEQ ID NO: 4. Includes No. MIMAT0015041) and other species homologs or orthologs. The hsa-miR-1260b gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-1260b" includes a sequence of "hsa-mir-1260b" (miRBase Accession No. MI0014197, SEQ ID NO: 13) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-486-5p gene" or "hsa-miR-486-5p" used as a determination miRNA herein refers to hsa-miR-486 consisting of the nucleotide sequence set forth in SEQ ID NO: 5. Includes -5p gene (miRBase Accession No. MIMAT0002177) and other species homologs or orthologs. The hsa-miR-486-5p gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-486-5p" includes a sequence of "hsa-mir-486-1" (miRBase Accession No. MI0002470, SEQ ID NO: 14) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-92a-3p gene" or "hsa-miR-92a-3p" used as a determination miRNA herein refers to hsa-miR-92a consisting of the nucleotide sequence set forth in SEQ ID NO: 6. Includes the -3p gene (miRBase Accession No. MIMAT0000092) and other species homologs or orthologs. The hsa-miR-92a-3p gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-92a-3p" includes a sequence of "hsa-mir-92a-1" (miRBase Accession No. MI00000903, SEQ ID NO: 15) having a hairpin-like structure as a precursor thereof.

In the present invention, the miRNA consisting of the nucleotide sequences represented by SEQ ID NOs: 7 to 9 described below can be used as a reference miRNA for determining hybridization.

The term "hsa-miR-149-3p gene" or "hsa-miR-149-3p" used as a reference miRNA herein refers to hsa-miR-149 consisting of the nucleotide sequence set forth in SEQ ID NO: 7. Includes the -3p gene (miRBase Accession No. MIMAT0004609) and other species homologs or orthologs. The hsa-miR-149-3p gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-149-3p" includes a sequence of "hsa-mir-149" (miRBase Accession No. MI0000478, SEQ ID NO: 16) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-2861 gene" or "hsa-miR-2861" used as a reference miRNA herein refers to the hsa-miR-2861 gene (miRBase Accession) consisting of the nucleotide sequence set forth in SEQ ID NO: 8. Includes No. MIMAT0013802) and other species homologs or orthologs. The hsa-miR-2861 gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-2861" includes a sequence of "hsa-mir-2861" (miRBase Accession No. MI0013006, SEQ ID NO: 17) having a hairpin-like structure as a precursor thereof.

The term "hsa-miR-4463 gene" or "hsa-miR-4463" used as a reference miRNA herein refers to the hsa-miR-4463 gene (miRBase Accession) consisting of the nucleotide sequence set forth in SEQ ID NO: 9. Includes No. MIMAT0018987) and other species homologs or orthologs. The hsa-miR-2861 gene can be obtained by the method described in Lagos-Quintana M et al., 2002, Curr Biol, Vol. 12, p735-739. Further, "hsa-miR-4463" includes a sequence of "hsa-mir-4463" (miRBase Accession No. MI0016811, SEQ ID NO: 18) having a hairpin-like structure as a precursor thereof.

Hereinafter, the process of selecting the determination miRNA used for determining the quality of the hybridization reaction of the present invention and the result of determining the quality of the hybridization reaction from the measurement data using the selected determination miRNA will be described. However, the present invention is not limited to the following examples.

Example 1
Selection of miRNA for determination (1) DNA chip The following experiment was carried out using "3D-Gene" human miRNA oligo chip (corresponding to miRBase release 22) manufactured by Toray Industries, Inc. This product is filled with microbeads for stirring in order to improve the stirring efficiency in the hybridization reaction step.

(2) Preparation and labeling of sample RNA Blood was collected from one healthy human and serum was prepared. RNA contained in a serum sample (hereinafter referred to as sample RNA) was extracted from the prepared serum using a “3D-Gene” RNA extraction reagent sample kit (Toray Industries, Inc.). The resulting sample RNA was labeled with a "3D-Gene" miRNA labering kit (Toray Industries, Inc.). At the time of labeling, an external standard nucleic acid was added to correct the measured value of miRNA to the expression level.

(3) Hybridization The labeled sample RNA was subjected to a hybridization reaction at 32 ° C. for 16 hours using a “3D-Gene” miRNA chip (Toray Industries, Inc.) as a DNA chip. The reaction is carried out under the condition that the DNA chip is swirled and stirred at 250 rpm using a bioshaker (Tietech Co., Ltd.) (hereinafter, referred to as “stirring condition” in Example 1), and the condition that the DNA chip is allowed to stand without swirling (hereinafter, carried out). In Example 1, it was carried out under two conditions (referred to as "standing condition").

(4) Measurement of Hybridization Amount The hybridized DNA chip was subjected to a microarray scanner (Toray Co., Ltd.), and the hybridization amount of each miRNA was measured. The scanner was set to 100% laser output, and the voltage setting of the photomultiplier was set to AUTO, and the fluorescence signal was measured.

The obtained fluorescence signal value was converted to a logarithm having a base of 2, and correction was performed with an external standard nucleic acid added at the time of labeling to obtain the hybridization amount of each miRNA.

As described above, the amount of hybridization of each miRNA under each hybridization condition was measured three times, and the average value thereof was obtained.

(5) Selection of miRNA for determination By comparing the miRNA expression levels under each hybridization condition obtained as described above, and extracting a miRNA having a large expression level that varies depending on the presence or absence of stirring during hybridization. , The determination miRNA of the present invention was selected.

First, from the detected miRNAs, 843 types of miRNAs were narrowed down as miRNAs that were stably detected in the high signal region (4.3 or higher).

Next, the difference between the hybridization amount of each miRNA under stirring conditions and the hybridization amount of each miRNA under standing conditions ((hybridization amount under stirring conditions)-(hybridization amount under standing conditions). ) Was obtained, and a miRNA having a difference of 2 or more in the amount of hybridization under these two conditions was selected as the determination miRNA. Table 1 shows the six types of determination miRNAs (SEQ ID NOs: 1 to 6) selected, the amount of hybridization thereof, and the difference between the above two conditions. Table 1 also shows the amount of hybridization of 843 types of miRNAs narrowed down and the average value of the differences between the above two conditions.

From the results in Table 1, the average value of the hybridization amount of all 843 miRNAs increased by 0.4 in the case of the stirring condition as compared with the case of the standing condition. It was confirmed that the stirring operation by the above gives the effect of improving the efficiency of the hybridization reaction. In addition, it was confirmed that the selected 6 types of miRNAs have a remarkable effect and can be used as a determination miRNA for determining the quality of the hybridization reaction.

(6) Creation of determination formula and setting of threshold value As shown in Equation 2C, the quality of the hybridization reaction is determined by representing the hybridization amount (e) of the determination miRNA and the hybridization amount of a plurality of reference miRNAs. The difference from (R) was obtained, and when the value exceeded the threshold value (t2), it was determined to be good.

As the determination miRNAs, 6 types of miRNAs shown in SEQ ID NOs: 1 to 6 selected above were selected. In addition, as reference miRNAs, three types of non-degraded endogenous miRNAs (hsa-miR-149-3p, has-miR-2861, has-miR-4463) shown in SEQ ID NOs: 7 to 9 were selected and hybridized. The average value of these three types of miRNA was used as the amount. Table 2 shows the difference (e-R) between the hybridization amount of each determination miRNA and the average value of the hybridization amounts of the three reference miRNAs under the stirring condition and the standing condition.

As shown in the results of Table 2, a clear difference was observed in the difference (e-R) in the above-mentioned hybridization amounts of all 6 types of miRNA between the stirring condition and the standing condition. From this result, it was confirmed that the quality of the hybridization reaction can be determined using the formula 2C. As the threshold value (t2) used for the determination, an intermediate value of the difference (e-R) between the stirring condition and the standing condition was set. The threshold value (t2) of each determination miRNA is shown in Table 2.

Example 2
Judgment of quality of hybridization reaction using judgment miRNA 1
(1) DNA chip The same DNA chip as in Example 1 was used.

(2) Preparation and labeling of sample RNA Blood was collected from three healthy humans, which was different from Example 1, and sample RNA was prepared by the same method as in Example 1 (1). The obtained sample RNA was designated as Specimen 1, Specimen 2, and Specimen 3 for each individual. The sample RNA was labeled in the same manner as in Example 1 (1).

(3) Hybridization The labeled sample RNAs (Samples 1 to 3) were subjected to a hybridization reaction at 32 ° C. for 16 hours using a “3D-Gene” miRNA chip (Toray Industries, Inc.). The reaction was carried out under the condition that the DNA chip was swirled and stirred at 250 rpm using a bioshaker (Tietech Co., Ltd.).

(4) Measurement of Hybridization Amount The hybridization amount of each miRNA was obtained by the same method as in Example 1 (4).

(5) Judgment of quality of hybridization reaction Six kinds of judgment miRNAs shown by SEQ ID NOs: 1 to 6 selected in Example 1 and three kinds of criteria shown by SEQ ID NOs: 7 to 9 used in Example 1 Using miRNA, the quality of the hybridization reaction was determined based on the amount of hybridization obtained in (4) above.

As shown in Equation 2C, the quality of the hybridization reaction is determined by the difference between the hybridization amount (e) and the average value (R) of the hybridization amounts of the three reference miRNAs for each determination miRNA. (E-R) is determined, and when the value exceeds the threshold value (t2) (Table 2) obtained in Example 1, it is determined to be good, and when it is below the threshold value, it is determined to be defective, and the determination miRNA determined to be good is determined. When the number was 4 or more, the hybridization reaction was comprehensively judged to be good. Table 3 shows the judgment results and the results of the comprehensive judgment for each judgment miRNA.

In all of the samples 1 to 3, the judgment that the hybridization reaction was good was obtained in all of the determination miRNAs, and the judgment that the hybridization reaction was good was also obtained in the comprehensive judgment.

In this example, in the hybridization step, the efficiency of the hybridization reaction is improved by stirring by swirling in a horizontal plane using stirring beads, and thus it is judged that this determination result is appropriate. Will be done.

Example 3
Judgment of quality of hybridization reaction using judgment miRNA 2
In the reaction step, the quality of the hybridization reaction was determined for Samples 1 to 3 in the same manner as in Example 2 except that the hybridization reaction was carried out with the DNA chip standing still. The results are shown in Table 4.

In all of the samples 1 to 3, it was determined that the hybridization reaction was poor in all of the determination miRNAs, and the comprehensive determination also determined that the hybridization reaction was poor.

In this example, since the DNA chip is left to stand in the hybridization step, the sample is not sufficiently stirred, and it can be considered that the efficiency of the hybridization reaction is lower than that in the case of stirring. Therefore, it is judged that this determination result is appropriate.

Example 4
Judgment of quality of hybridization reaction using judgment miRNA 3
Samples 1 to 3 were the same as in Example 2 except that the reaction step was carried out under the condition that the DNA chip was swirled and stirred at 250 rpm using a bioshaker (Tietech Co., Ltd.) without using stirring beads. The quality of the hybridization reaction was determined. The results are shown in Table 5.

In the case of Samples 1 and 2, it was determined that the hybridization reaction was poor in all of the determination miRNAs, and it was also determined that the hybridization reaction was poor in the comprehensive determination. In the case of Specimen 3, two of the determination miRNAs were judged to have a good hybridization reaction, and four types were judged to have a poor hybridization reaction. As a result, it was determined that the hybridization reaction was poor in the comprehensive judgment.

In this measurement, in the hybridization step, the DNA chip is swirled in a horizontal plane to be stirred without using stirring beads, so that the sample is not sufficiently stirred, and the efficiency of the hybridization reaction is higher than that in the case of stirring. Is considered to be decreasing, so it is judged that this judgment result is appropriate.

Example 5
Judgment of quality of hybridization reaction using judgment miRNA 4
The quality of the hybridization reaction for Samples 1 to 3 was the same as in Example 2 except that the DNA chip was swirled and stirred at 12 rpm using a microtube rotator (AS ONE Corporation) in the reaction step. Was judged. The results are shown in Table 6.

In all of the samples 1 to 3, the judgment that the hybridization reaction was good was obtained in all of the determination miRNAs, and the judgment that the hybridization reaction was good was also obtained in the comprehensive judgment.

In this embodiment, in the hybridization step, the efficiency of the hybridization reaction is improved by swirling on a vertical plane (a plane parallel to the direction of gravity) using stirring beads to perform stirring. This judgment result is judged to be valid.

Claims (7)

A method for determining the quality of the hybridization reaction of the nucleic acid to be detected in the detection of nucleic acid in a sample using a DNA chip.
DNA on which one or more determination miRNAs selected from miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6 and a probe that hybridizes with one or more predetermined reference miRNAs are immobilized on the substrate. A reaction step in which a sample is brought into contact with the chip to carry out a hybridization reaction;
A measurement step of measuring the hybridization amount of one or more determination miRNAs obtained in the reaction step and the hybridization amount of one or more reference miRNAs; and one or more determinations obtained in the measurement step. A determination step of comparing the hybridization amount of the target miRNA with the hybridization amount of one or more reference miRNAs to determine the quality of the hybridization reaction of the nucleic acid to be detected;
How to include. The method according to claim 1, wherein the sample is agitated using fine particles or bubbles in the reaction step. The method according to claim 2, wherein in the reaction step, the sample is agitated by moving the fine particles by gravity or centrifugal force. In the determination step, the hybridization reaction is good when the difference or ratio between the hybridization amount of one or more determination miRNAs and the hybridization amount of one or more reference miRNAs exceeds a predetermined threshold as a reference. The method according to any one of claims 1 to 3. The method according to any one of claims 1 to 4, wherein the nucleic acid to be detected is miRNA. The method according to any one of claims 1 to 5, wherein the sample is blood, serum or plasma. A DNA chip for quality determination of the hybridization reaction of the nucleic acid to be detected.
A probe for capturing a quality-determining miRNA for one or more hybridization reactions selected from miRNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6, and a probe for capturing one or more reference miRNAs. A DNA chip in which a probe is immobilized on a substrate.