JP2009118774A - Method for detecting rna sequence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting RNA sequence by which a plurality of target genes can be detected simply and quickly. <P>SOLUTION: The method for detecting the RNA sequences includes (a) a step for using a carrier having continuous identical reaction spaces, a first region to which an oligonucleotide for catching RNA is immobilized, and a second region to which two or more oligonucleotides for detecting the RNA sequences are immobilized, supplying a solution containing the RNA of a detection target to the reaction spaces, and hybridizing the RNA with the immobilized oligonucleotide for catching the RNA to catch the RNA; (b) a step for elongating the oligonucleotide for catching the RNA with the caught RNA as a template to synthesize a cDNA chain; (c) a step for amplifying the DNA chain with the cDNA chain as a template; and (d) a step for detecting the state of the hybridization of the amplified DNA chain with the immobilized oligonucleotide for detecting the RNA sequence. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an RNA detection method for quantitatively and qualitatively detecting a plurality of RNA strands in a sample using a carrier having a primer DNA strand immobilized on the surface.

  Detection and diagnosis using genes such as gene expression status and identification of bacteria and viruses are routinely performed in the field of biochemistry. Conventionally, detection of genes has been performed by electrophoresis, but recently, a method of simultaneously viewing a plurality of genes using a DNA microarray has been performed. It is possible to qualitatively and quantitatively analyze a plurality of genes by amplifying and detecting a target gene sequence even with a small amount of RNA in a sample. In the conventional method using electrophoresis, it takes time to detect a reaction by PCR, electrophoresis time, etc., and the operation of electrophoresis is troublesome. However, it has become possible to simultaneously analyze a plurality of genes by DNA microfluidics.

  Currently, as a method for detecting a gene sequence using a DNA microarray, a method is used in which a probe DNA strand is immobilized on the surface of a predetermined carrier and a labeled DNA strand is hybridized and detected. Recently, a method has been devised in which a gene is detected by extending an extension primer immobilized on the surface of a carrier using a DNA fragment as a template. As one of them, Non-Patent Document 1 discloses that a DNA strand serving as a primer is covalently bonded to the surface of a glass substrate modified with a predetermined amino-silane reagent, and a PCR reaction is performed on the substrate to perform DNA amplification. Techniques to do are disclosed. Non-Patent Document 2 discloses that a DNA microarray in which a DNA fragment is immobilized on a poly (methyl methacrylate) surface instead of a glass substrate is used for hybrid characteristics with a predetermined DNA strand and thermal stability under PCR reaction conditions. There is a description that suggests the possibility of a device that can be applied to a novel PCR technique.

In any method using a DNA microarray, operations such as RNA extraction and purification from a biological sample, reverse transcription reaction, amplification reaction, and detection by a DNA microarray are very complicated, and there are problems such as sample loss and contamination in each process. In order to perform stable quantification of gene expression level, it is necessary to have a skill of a researcher.
Adessi, Celine et al. “Solid Phase DNA amplification: Characterization of primer attachment and amplification mechanisms”, Nucleic Acids Research, 2000, Vol. 20, No. 20, e87 Fixe, F. et al. “Functionalization of poly (methyl-5-methacrylate) (PMMA) as a substrate for DNA microarrays”, Nucleic Acids Research, 2004, January 12, Vol.32 No.1, e9

  An object of the present invention is to provide a method for detecting an RNA sequence that is easy to operate and detects a plurality of target genes rapidly. The present inventors have completed the present invention by performing RNA extraction, amplification reaction, and detection in the same reaction space.

The present invention is as follows.
(1) A method for detecting a plurality of RNA sequences, the same continuous reaction space, a first region in which an RNA capturing oligonucleotide for capturing RNA is immobilized, and a homology with an RNA sequence to be detected Using a carrier having a second region in which two or more kinds of oligonucleotides for RNA sequence detection having a sequence to be immobilized are immobilized,
(A) supplying a solution containing RNA to be detected to the reaction space, hybridizing the RNA and the immobilized RNA capturing oligonucleotide, and capturing the RNA;
(B) synthesizing a cDNA strand by extending an immobilized RNA capturing oligonucleotide using the captured RNA as a template;
(C) a step of amplifying a DNA strand using the cDNA strand synthesized in (b) as a template,
(D) detecting the state of hybridization between the amplified DNA strand and the immobilized RNA sequence detection oligonucleotide;
A method for detecting an RNA sequence comprising
(2) The method for detecting an RNA sequence according to (1), wherein the RNA capturing oligonucleotide is a dT sequence.
(3) The method for detecting an RNA sequence according to (1) or (2), wherein the RNA captured in step (a) is mRNA.
(4) The method for detecting an RNA sequence according to any one of (1) to (3), wherein the amplification of the DNA strand in step (c) is any one of PCR, LCR, SDA, RCA, TMA, ICA, NASBA, and TRC .
(5) The method for detecting an RNA sequence according to any one of (1) to (4), wherein the detection of the hybridization status in step (d) is fluorescence detection using a fluorescence intercalator.
(6) The amplification of the DNA strand in the step (c) is PCR, a label is introduced into the DNA strand to be amplified, and the label introduced in the step (d) is detected (1) to (4) Method for detecting RNA sequence of
(7) The method for detecting an RNA sequence according to (6), wherein in step (c), a labeled mononucleotide is added to the PCR reaction system.
(8) The method for detecting an RNA sequence according to any one of (1) to (7), wherein the first region and the second region are clearly distinguished.
(9) The shape of the carrier is one selected from a glass slide shape, a film shape, a microtiter plate shape, a PCR tube shape, and a PCR multiwell shape (1) to (8) A method for detecting an RNA sequence according to claim 1.
(10) The method for detecting an RNA sequence according to any one of (1) to (9), wherein the reaction space shape is one selected from a plane, a well, a container, and a fine channel.
(11) The method for detecting an RNA sequence according to any one of (1) to (10), wherein the first region and the second region are sequentially formed in the flow path in the carrier.
(12) The method for detecting an RNA sequence according to any one of (1) to (11), wherein the surface of the carrier is hydrophilic.
(13) The method for detecting an RNA sequence according to any one of (1) to (12), wherein the carrier surface has a group derived from a phospholipid ester constituting a hydrophilic part of a phospholipid.

  According to the present invention, the operation is simple and a plurality of target RNAs can be detected quickly.

  Hereinafter, embodiments of the present invention will be described in detail.

  The present invention is a method for detecting a plurality of RNA sequences, the same continuous reaction space, a first region in which an RNA capture oligonucleotide for capturing RNA is immobilized, and a homology with an RNA sequence to be detected The solution containing the RNA to be detected was supplied to the reaction space using the carrier having the second region to which each of two or more kinds of RNA sequence detection oligonucleotides having the sequence to be immobilized was immobilized, and the RNA was immobilized A step of hybridizing with an RNA capture oligonucleotide to capture the RNA, a step of extending an immobilized RNA capture oligonucleotide using the captured RNA as a template to synthesize a cDNA strand, a cDNA strand as a template, Step of amplifying the DNA strand, hybridization of the amplified DNA strand and the immobilized oligonucleotide for detecting the RNA sequence A step of detecting a method for detecting RNA sequences containing.

  The reaction space used in the present invention has, for example, a region in which an oligonucleotide for capturing RNA is immobilized in the same continuous reaction space as shown in FIG. 1 and a sequence homologous to the RNA sequence to be detected 2. It has a plurality of regions in which more than one type of RNA sequence detection oligonucleotide is immobilized.

  Biological components such as proteins contained in the cell lysate inhibit the enzyme reaction of the reverse transcription reaction and the amplification reaction. In addition, the enzyme itself used for the enzyme reaction is adsorbed on the inner wall of the reaction space to inhibit the reactivity. Therefore, in the present invention, in order to capture and purify RNA from the cell lysate, it is desirable that the surface of the carrier has hydrophilicity and has the property of suppressing the adsorption of proteins and nucleic acids and not inhibiting the enzymatic reaction. . The carrier surface must have a functional group for immobilizing an oligonucleotide for capturing or detecting RNA.

  For example, a first unit having a group derived from a phosphate ester constituting the hydrophilic part of a phospholipid and a second unit having a carboxylic acid derivative group in which an electron withdrawing substituent is bonded to a carbonyl group. Examples thereof include a carrier having a polymer substance contained on the surface.

  Hereinafter, this polymer substance will be described.

  It has a first unit containing a group derived from a phosphate ester constituting the hydrophilic part of this phospholipid and a second unit having a carboxylic acid derivative group in which an electron-withdrawing substituent is bonded to a carbonyl group. The polymer substance is a polymer having both the property of suppressing nonspecific adsorption of DNA strands and the property of immobilizing DNA strands. In particular, a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid contained in the first unit plays a role in suppressing nonspecific adsorption of the template DNA fragment, and the carboxylic acid-derived group contained in the second unit. Serves to chemically immobilize the primer. That is, the primer is covalently bonded at the site of the carboxylic acid derivative group of the coating layer made of the polymer material and immobilized on the surface.

The first unit is, for example, a (meth) acryloyloxyalkyl phosphorylcholine group such as a 2-methacryloyloxyethyl phosphorylcholine group, a 6-methacryloyloxyhexyl phosphorylcholine group;
(Meth) acryloyloxyalkoxyalkylphosphorylcholine groups such as 2-methacryloyloxyethoxyethyl phosphorylcholine group and 10-methacryloyloxyethoxynonylphosphorylcholine group;
Alkenylphosphorylcholine groups such as allylphosphorylcholine group, butenylphosphorylcholine group, hexenylphosphorylcholine group, octenylphosphorylcholine group, and decenylphosphorylcholine group;
And a phosphorylcholine group is included in these groups.

  Of these groups, 2-methacryloyloxyethyl phosphorylcholine is preferable. By adopting a configuration in which the first unit has 2-methacryloyloxyethyl phosphorylcholine, nonspecific adsorption of a biological component on the surface can be more reliably suppressed.

  The carboxylic acid derivative is a carboxylic acid in which the carboxyl group of the carboxylic acid is activated and has a leaving group via C═O. Specifically, the carboxylic acid derivative is a compound in which a nucleophilic reaction is activated by bonding a group having higher electron withdrawing property than an alkoxyl group to a carbonyl group. The carboxylic acid derivative group is a compound having reactivity with an amino group, a thiol group, a hydroxyl group and the like.

  More specifically, as the activated carboxylic acid derivative, carboxyl groups such as acrylic acid, methacrylic acid, crotonic acid, maleic acid, and fumaric acid, which are carboxylic acids, are acid anhydrides, acid halides, active esters, Examples include compounds converted to activated amides. Carboxylic acid-derived groups are activated groups derived from such compounds, for example, active ester groups such as p-nitrophenyl and N-hydroxysuccinimide groups, and groups such as halogens such as —Cl and —F. Can have.

  Of the carboxylic acid-derived groups, an active ester group is preferably used because of its excellent reactivity under mild conditions. The mild conditions include, for example, neutral or alkaline conditions, specifically pH 7.0 or more and 10.0 or less, more specifically pH 7.6 or more and 9.0 or less, and more specifically pH 8.0. It can be.

  In addition, the polymer substance used for the coating layer of the carrier of this embodiment may contain other groups in addition to the phosphorylcholine group and the carboxylic acid derivative group. The polymer substance can be a copolymer. Specifically, the polymer substance is preferably a copolymer containing a butyl methacrylate group. By so doing, the polymer substance can be appropriately hydrophobized, and the adsorptivity of the polymer substance to the carrier surface can be more suitably ensured.

  Specifically, the polymer substance is composed of a first monomer having a 2-methacryloyloxyethyl phosphorylcholine (MPC) group and a second monomer having a p-nitrophenyloxycarbonyl polyethylene glycol methacrylate (NPMA) group. , And a copolymer with a third monomer having a butyl metallate (BMA) group. These copolymers, poly (MPC-co-BMA-co-NPMA) (PMBN), are schematically represented by the following general formula (1).

  However, in the said General formula (1), a, b, and c are respectively independently a positive integer. In the general formula (1), the first to third monomers may be block copolymerized, or these monomers may be copolymerized randomly.

  The copolymer represented by the general formula (1) is more suitable for the balance between moderate hydrophobicity of the polymer substance, the property of suppressing non-specific adsorption of the template RNA fragment, and the property of immobilizing the primer. It is an even better configuration. For this reason, by using such a copolymer, the surface of the carrier is more reliably coated with the polymer material, and nonspecific adsorption of the template RNA fragment onto the carrier coated with the polymer material is suppressed. However, the primer can be more reliably immobilized by covalent bonding and introduced onto the carrier.

  The first unit of the first polymer substance and the first unit of the second polymer substance may have the same structure or different structures. In addition, when the first polymer substance includes a third unit containing a butyl methacrylate group, the third unit of the first polymer substance and the third unit of the second polymer substance have the same structure. There may be a different structure.

  Such a second polymer substance is used as a polymer that suppresses nonspecific adsorption of the template DNA fragment. As such a polymer, for example, an MPC polymer (manufactured by NOF Corporation) containing 30 mol% phosphorylcholine groups and 70 mol% butyl methacrylate groups can be used.

  The carrier including the coating layer made of the polymer material as described above can be obtained by applying a liquid containing the polymer material on the surface of the carrier processed into a predetermined shape and drying it. Alternatively, the carrier may be immersed in a liquid containing a polymer substance and dried.

  Further, when a plastic material is used as the carrier, flexibility in changing the shape and size is ensured, and it is preferable from the viewpoint that it can be provided at a lower cost than that of the glass carrier. As such a plastic material, a thermoplastic resin can be used from the viewpoint of easy surface treatment and mass productivity.

  The thermoplastic resin is not particularly limited as long as it has a certain degree of heat resistance. Examples of the heat resistant resin include linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, fluorine-containing resins, and the like.

  When the support is exposed to a high temperature close to 100 ° C. as a reaction condition in detection, polypropylene or cyclic polyolefin is more preferable from the viewpoint of thermal stability.

Next, a method for immobilizing a primer (an RNA capturing oligonucleotide for capturing RNA) on the surface of the carrier will be described.
For example, (i) out of a plurality of active ester groups contained in the polymer substance on the carrier, at least a part of the active ester groups react with the primer to form a covalent bond, thereby fixing the primer on the surface of the carrier. Followed by (ii) inactivating the active ester group on the surface of the carrier other than the one on which the primer is immobilized, i.e. inactivating the remaining active ester groups, thereby immobilizing the primer on the surface of the carrier. it can. Hereinafter, each process will be described.

  In the above step (i), when immobilizing the primer to be annealed with the template RNA fragment on the carrier, a part of the active ester group contained in the polymer substance is brought into contact with the liquid in which the primer is dissolved or dispersed. Reacts with the primer to form a covalent bond between the primers.

  The liquid in which the primer is dissolved or dispersed can be, for example, neutral to alkaline, for example, pH 7.6 or higher.

  Further, after the contact, in order to remove the primer not immobilized on the surface of the carrier, it may be washed with pure water or a buffer.

  In addition, an amino group is preferably introduced into the primer to be immobilized on the carrier in order to increase the reactivity with the active ester group. Since the amino group is excellent in reactivity with the active ester group, the primer can be efficiently and firmly immobilized on the surface of the carrier by using the primer into which the amino group has been introduced. The amino group may be introduced at the end of the molecular chain or at the side chain of the primer, but it can be annealed with the complementary template RNA fragment more efficiently if it is introduced at the end of the molecular chain. From this point of view, it is preferable.

When the carrier material is plastic, the shape is not limited to a plate shape, the shape of a microtiter plate typified by 96 or 384 holes, the shape of a substrate typified by a slide glass, or the shape of a PCR tube. The shape of a thing, a sheet form, a film form, or a fine channel is raised.

  The carrier on which the oligonucleotide obtained as described above is immobilized has nonspecific adsorption on the carrier surface suppressed by the effect of the hydrophilic group of the polymer, compared to a general plastic surface. For this reason, for example, RNA complementary to the primer immobilized on the surface is captured from a sample mixed with biological components such as cell lysate, and the supernatant is removed and washed to purify the captured RNA. be able to.

  An oligonucleotide to be immobilized for detection can detect a plurality of sequences by spot immobilization.

Hereinafter, specific embodiments of the RNA detection method using this carrier will be described. About the following embodiment, it illustrates about the case where the solution containing RNA of detection object is a sample containing a cell lysis solution.
(First embodiment)
FIG. 1 shows an example of a reaction space obtained from the carrier. It consists of an oligonucleotide part (RNA capture part) for capturing RNA and an oligonucleotide part (detection part) for detection in the same reaction space. FIG. 2 is a flowchart showing the procedure of the RNA detection method as an embodiment. 3 and 4 are diagrams schematically showing the reaction performed in the reaction space of the carrier according to the flowchart of FIG.

  In this RNA detection method, a sample 22 containing a cell lysate is introduced into a reaction space 20 provided in the carrier 12 (step S10), and hybridization is performed (step S20). First step and washing are performed (step S30). In the second stage, a sample 23 containing an enzyme system for DNA chain extension and nucleotide monomers is introduced (step S40), and the RNA strand 16 contained in the cell lysate is used as a template for the oligonucleotide for RNA capture (primer 14). Performing a DNA chain extension reaction to form a cDNA chain 18 (step S50), and a reaction system including the cDNA chain 18 in an extension DNA primer, an enzyme system for DNA chain extension, a nucleotide monomer, and a labeled nucleotide monomer (Step S60), using the cDNA strand 18 as a template, P When the R reaction is performed and the labeled DNA strand 21 is amplified (step S70), the amplified DNA 21 is hybridized to the RNA sequence detection oligonucleotide (primer 15) immobilized in the reaction space, and DNA The strand 21 is used as a template to perform a DNA strand elongation reaction of the RNA sequence detection oligonucleotide to produce a labeled DNA strand 23 (step S80), and the fourth stage is detected using the label (step S90). Stages.

When the cell lysate introduced in step S10 does not inhibit the enzyme reaction by the enzyme system for DNA chain extension, or when there is a device for reducing the inhibition of the enzyme reaction, the sample 22 containing the cell lysate is subjected to DNA chain extension. Enzyme systems and oligonucleotide monomers can be added and steps S20 and S30 can be omitted.

In step S20, the target RNA strand 16 in the cell lysate is hybridized with the immobilized primer 14, and the target RNA strand 16 is captured. (FIGS. 3B and 4B) RNA strands that are not complementary to the sequence of the immobilized primer 14 cannot hybridize and exist in the liquid phase. By using an oligo dT primer as the primer 14, it is possible to capture mRNA by hybridization with poly A which is generally possessed by mRNA. Further, by using a DNA primer having a specific sequence in the primer 14 other than poly A, only a complementary RNA strand can be captured.

  In step S30, by washing the reaction space 20, the sample 22 in the reaction space can be removed leaving the double strands of the primer 14 and the RNA strand 16. The surface coated with the polymer in the washing can be purified as a pure RNA strand 16 because the hydrophilic group has a non-adsorption effect on the biomolecule contained in the cell lysate. When a plastic surface such as polystyrene or polypropylene having low non-adsorption property is used on the surface, an enzyme reaction inhibitor in a subsequent process cannot be removed, which adversely affects detection.

  In step S40, an enzyme reaction system used for the reverse transcription reaction is introduced. For example, Reverse Transcriptase M-MLV (Rnase H-), rTth polymerase, etc. are raised as an enzyme having reverse transcription reaction ability.

  In step S50, a reverse transcription reaction takes place by the action of the enzyme system for DNA chain elongation using the RNA chain 16 as a template at the 3 ′ end of the primer 14 to produce a DNA chain. A cDNA strand 18 complementary to the RNA strand 16 is formed on the substrate 12. (FIG. 3C, FIG. 4C) Moreover, when a primer having a sequence complementary to the RNA strand 16 is present in the liquid phase, the primer hybridizes with the RNA strand 16 to cause a reverse transcription reaction.

  In step S60, a PCR reagent including a primer (not shown) having a sequence complementary to a part of the cDNA strand 18 using the cDNA strand 18 as a template DNA strand, a DNA strand extender system, a nucleotide monomer, and a labeled nucleotide monomer is prepared. Introduce. Examples of the DNA chain elongation enzyme system include thermostable enzymes such as Taq polymerase typically used in PCR.

  In step S70, a DNA amplification reaction occurs by the action of the cDNA strand 18 and a primer having a sequence complementary to a part of the cDNA strand 18 and the DNA strand elongation enzyme system, and the DNA strand 21 is amplified (FIGS. 3D and 4D). ). At this time, if a labeled nucleotide monomer is contained in step S60, the DNA strand 21 is labeled.

  In step 80, the DNA strand 21 produced by the amplification reaction is hybridized with an oligonucleotide (probe 15) having a complementary sequence immobilized in the reaction space and captured. At this time, at the 3 ′ end of the primer 15, a DNA strand elongation reaction occurs by the action of the DNA strand elongation enzyme system using the DNA strand 21 as a template, and a DNA strand 23 complementary to the DNA strand 21 is formed on the substrate 12. The At this time, if a labeled nucleotide monomer is contained in step S60, the DNA strand 23 is labeled.

  In step S90, the DNA strand 21 labeled in step 80 and the solid-phased DNA strand 23 can be detected using a label. By using a fluorescent substance for labeling, it can be detected with a fluorescent microscope, a fluorescent scanner, or the like. Moreover, it can detect with high sensitivity by using biotin as a label and binding an enzyme such as avidin-immobilized alkaline phosphatase. Quantification can be performed by digitizing the detected signal.

  In step S40 to step S80, step S60 is omitted by using a one-step type RT-PCR reaction solution in which reverse transcriptase for preparing cDNA and DNA polymerase for performing PCR reaction can be performed in the same reaction solution. can do.

(Preparation of PMBN coat reaction space)
A reaction space as shown in FIG. 1 was produced by cutting a plastic plate. This reaction space was divided into 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate-p-nitrophenyloxycarbonyl polyethylene glycol methacrylate copolymer (poly (MPC-co-BMA-co-NPMA) (PMBN), each group in mol%). A polymer substance having a phosphorylcholine group and an active ester group on its surface was introduced by immersing it in a 0.5 wt% ethanol solution of 25: 74: 1) to obtain a carrier coated with PMBN. Hereinafter, it is referred to as a PMBN coated carrier.

(Primer fixation)
As primers, oligo DNAs whose 5 ′ ends were modified with amino groups as shown in Table 1 were each dissolved using 0.25 M carbonate buffer (pH 9.0) to prepare 10 μM oligo DNA solutions. The oligo DNA solution of the capture primer was immobilized on the PMBN coated carrier as shown in FIG. Similarly, the oligo DNA solution of the detection primer was spotted in a spot shape as shown in FIG. 1, and the carrier was allowed to stand at 80 ° C. for 1 hour to be immobilized on the carrier. After washing with ultrapure water, a blocking treatment was performed.

  This blocking treatment is to immobilize oligo DNA on the surface of the carrier and use it as an immobilized primer, then inactivate the unreacted active ester present on the surface of the carrier, and the DNA and RNA strands in the sample, Other treatments for preventing non-specific binding of biological substances such as proteins.

  Specifically, with respect to the PMBN coated carrier, 400 μl of 1 mol / l NaOH solution as a blocking solution was dispensed into the reaction space and left at room temperature for 5 minutes, and then the blocking solution was removed and washed with ultrapure water. .

  After blocking treatment, 400 μl of 1% BSA was dispensed into the reaction space in PBS (−), left at room temperature for 2 hours, and 400 μl of a washing solution containing Tween 20 at a concentration of 0.05% in PBS (−) Poured and removed by suction three times, and then dried.

(Purification of RNA from rat tissue)
Adipocytes of normal rats and hypertensive obese rats were prepared, a cell lysate containing guanidine was added to each cell, and the cells were stirred well by pipetting or the like to obtain a cell lysate of rat adipocytes. Diluted solutions of these solutions were dispensed in a separate PMBN-coated carrier at 50 μl and allowed to stand at room temperature for 15 minutes. After standing, 200 μl of a buffer solution containing a surfactant was dispensed, and suction was repeated and washed twice. MRNA having poly A is captured by the dT primer on the surface of the carrier, and biological substances other than mRNA contained in the cell lysate can be removed.

(Synthesis of solid phase cDNA from mRNA)
The reaction solution was prepared by adding Reverse Transcriptase M-MLV (RNAse H-), 5 × Reverse Transcriptase M-MLV Buffer, RNAse Inhibitor (Super), 20 mM dTTP, 20 mM dATP, 20 mM dGTP, 20 mM dCTP, DEPC treated water. . 50 μl of this solution was dispensed onto each carrier and incubated at 42 ° C. for 15 minutes or longer. After incubation, the solution was removed.

(Solid phase cDNA to PCR reaction)
The reaction solution was prepared by adding ExTaq HS (manufactured by Takara Bio), 10 × ExTaq Buffer, 10 μM PCR Forward Primer, 10 μM PCR Reverse Primer, 20 mM Biotinylated dUTP, 20 mM dATP, 20 mM dGTP, 20 mM dCTP, DEPC treated water. 50 μl was dispensed onto a carrier and PCR was performed. At this time, an extension reaction of the amplified PCR product and the detection immobilized primer occurs and is labeled. Rat-derived β Actin and adrenergic receptor β3 were selected as the Forward primer and Reverse primer. The primer sequences are shown in Table 2.

(Coloring reaction)
After the extension reaction, the DNA extension reaction solution is removed and washed, and an alkaline phosphatase solution labeled with streptavidin is supplied to the substrate surface. After leaving at 37 ° C. for 30 minutes, the alkaline phosphatase solution is removed, After washing, an NBT / BICP solution was supplied, and the mixture was allowed to stand at 37 ° C. for 30 minutes to carry out an enzyme reaction. After the substrate was washed, the spotted portion was clearly spotted on the surface of the PNBM coated carrier. A colored image of the primer spot on the substrate was captured by a CCD camera, and the captured digital data was processed by image processing software (NIH image) to quantify the degree of coloring. The results are shown in Table 3.

The signal value obtained in a spot shape indicates a value depending on the amount of PCR product, and indicates the starting template amount in PCR. By obtaining the signal value, the expression level of the target RNA contained in the cell lysate can be analyzed. By arranging a plurality of spots in the same reaction space, multiple specimens can be examined.

It is a figure which shows typically one Embodiment of the support | carrier used for this invention. It is a flowchart which shows one Embodiment of the RNA detection method of this invention. It is a figure which shows typically the reaction performed in the reaction space of a support | carrier according to the flowchart of FIG. It is a figure which shows typically the reaction performed in the reaction space of a support | carrier according to the flowchart of FIG.

Explanation of symbols

12 Carrier 14 RNA capture oligonucleotide 15 RNA sequence detection oligonucleotide 16 RNA strand 18 cDNA strand 20 Reaction space 21 Labeled DNA strand 22 Sample containing cell lysate

Claims (13)

A method for detecting a plurality of RNA sequences, comprising: an identical continuous reaction space; a first region in which an RNA capturing oligonucleotide for capturing RNA is immobilized; and a sequence homologous to an RNA sequence to be detected. Using a carrier having a second region in which two or more kinds of oligonucleotides for RNA sequence detection having each immobilized thereon are used,
(A) supplying a solution containing RNA to be detected to the reaction space, hybridizing the RNA and the immobilized RNA capturing oligonucleotide, and capturing the RNA;
(B) synthesizing a cDNA strand by extending an immobilized RNA capturing oligonucleotide using the captured RNA as a template;
(C) a step of amplifying a DNA strand using the cDNA strand synthesized in (b) as a template,
(D) detecting the state of hybridization between the amplified DNA strand and the immobilized RNA sequence detection oligonucleotide;
A method for detecting an RNA sequence comprising The method for detecting an RNA sequence according to claim 1, wherein the oligonucleotide for RNA capture is a dT sequence. The method for detecting an RNA sequence according to claim 1 or 2, wherein the RNA captured in step (a) is mRNA. The method for detecting an RNA sequence according to any one of claims 1 to 3, wherein the amplification of the DNA strand in step (c) is any one of PCR, LCR, SDA, RCA, TMA, ICA, NASBA, and TRC. The method for detecting an RNA sequence according to any one of claims 1 to 4, wherein the detection of the hybridization status in the step (d) is fluorescence detection using a fluorescence intercalator. The detection of the RNA sequence according to any one of claims 1 to 4, wherein the amplification of the DNA strand in step (c) is PCR, a label is introduced into the DNA strand to be amplified, and the label introduced in step (d) is detected. Method. The method for detecting an RNA sequence according to claim 6, wherein in step (c), a labeled mononucleotide is added to the PCR reaction system. The method for detecting an RNA sequence according to any one of claims 1 to 7, wherein the first region and the second region are clearly distinguished. The RNA sequence according to any one of claims 1 to 8, wherein the shape of the carrier is one selected from a glass slide shape, a film shape, a microtiter plate shape, a PCR tube shape, and a PCR multiwell shape. Detection method. The method for detecting an RNA sequence according to any one of claims 1 to 9, wherein the reaction space shape is one selected from a plane, a well, a container, and a fine channel. The method for detecting an RNA sequence according to any one of claims 1 to 10, wherein in the carrier, a first region and a second region are sequentially formed in a flow path. The method for detecting an RNA sequence according to any one of claims 1 to 11, wherein the surface of the carrier is hydrophilic. The method for detecting an RNA sequence according to any one of claims 1 to 12, wherein the carrier surface has a group derived from a phospholipid ester constituting a hydrophilic part of a phospholipid.

Images