阅读说明：本技术 一种RNA甲基化m6A检测方法及试剂盒 (RNA methylation m6A detection method and kit ) 是由 林锦梅 陈少锐 于 2020-07-17 设计创作，主要内容包括：本发明涉及一种RNA甲基化m6A检测方法及试剂盒。本发明的检测方法采用抗体捕获和RNA逆转录相结合的方法,利用逆转录过程中以RNA为模板合成DNA的过程,针对目标基因m6A位点区域,设计特异寡核苷酸引物并偶连生物素检测指定基因m6A水平,及随机引物检测总RNAm6A水平。利用被m6A抗体捕获的RNA片段结合,并用带有HRP标记的链霉亲和素结合生物素进行二级信号放大,通过TMB显色检测吸光度并计算得到目标基因m6A的水平。本发明首次提供了一种不完全依赖抗体免疫酶联的系统性检测m6A的方法,具有高效、灵敏等优点,同时检测成本低、操作简便、适用于多种RNA样本,解决了传统检测方法分辨率有限、无法进行m6A定量、RNA样本需求量大的问题。(The invention relates to a method and a kit for detecting RNA methylation m 6A. The detection method of the invention adopts a method combining antibody capture and RNA reverse transcription, utilizes the process of synthesizing DNA by taking RNA as a template in the reverse transcription process, designs specific oligonucleotide primers aiming at the site region of the target gene m6A and couples biotin to detect the level of the designated gene m6A, and detects the level of total RNAm6A by random primers. The level of the target gene m6A was calculated by detecting absorbance through TMB color development using RNA fragment binding captured by the m6A antibody and secondary signal amplification using streptavidin-conjugated biotin with HRP label. The invention provides a method for systematically detecting m6A without completely depending on antibody immunoenzyme coupling for the first time, which has the advantages of high efficiency, sensitivity and the like, is low in detection cost, simple and convenient to operate, is suitable for multiple RNA samples, and solves the problems that the traditional detection method is limited in resolution, cannot carry out m6A quantification and is large in RNA sample requirement.)

1. A method for detecting RNA methylation m6A is characterized by comprising the following steps:

(1) adding the RNA sample into the experiment hole coated by the antibody, and incubating at room temperature;

(2) discarding liquid in the experimental hole, adding washing liquid, blowing and beating by using a liquid transfer gun, discarding the washing liquid, and repeating for three times;

(3) adding a reverse transcription primer into an experimental hole to perform reverse transcription reaction;

(4) adding streptavidin with a label into the experimental hole, and carrying out a binding reaction between the streptavidin and biotin;

(5) discarding liquid in the experimental hole, adding washing liquid, blowing and beating by using a liquid transfer gun, discarding the washing liquid, and repeating for three times;

(6) adding a color developing agent into the experimental hole, incubating, and adding a stop solution;

(7) the absorbance of the mixture in the test well was measured and calculated to give the m6A content.

2. The detection method according to claim 1, wherein the antibody is a m6A recombinant antibody.

3. The detection method according to claim 1, wherein the reverse transcription primer is at least one of Oligo dT, a random primer, and a gene-specific primer.

4. The detection method according to claim 1, wherein the label is horseradish peroxidase.

5. The detection method according to claim 1, wherein the incubation temperature is 20-25 ℃ and the incubation time is 1 h.

6. The detection method according to claim 1, wherein the detection wavelength of the absorbance is 450 nm.

7. An RNA methylation m6A detection kit, which is characterized by comprising the following reagents: washing liquid, sample extracting solution, reverse transcription reagent, negative control liquid, positive control liquid, horseradish peroxidase labeled avidin and stop solution.

8. The detection kit according to claim 7, wherein the washing solution is at least one of sodium chloride, potassium chloride, tris (hydroxymethyl) aminomethane and tween 20;

the sample extracting solution is at least one of phenol, guanidinium isothiocyanate and beta-mercaptoethanol;

the reverse transcription reagent is at least one of random primer labeled biotin, target gene primer labeled biotin, polythymidine, T repeated oligonucleotide, deoxyribonucleoside triphosphate, RNase inhibitor and DNA reverse transcription polymerase;

the concentration of RNA without m6A in the negative control solution is 100 mug/mL;

the concentration of the m6A oligonucleotide in the positive control solution is 2 mug/mL;

the concentration of the sulfuric acid in the stop solution is 2 mol/L.

9. The detection kit of claim 7, further comprising a dilution of horseradish peroxidase-labeled avidin and a substrate solution.

10. The detection kit of claim 9, wherein the horseradish peroxidase labeled avidin diluent is a balanced salt solution;

the substrate solution comprises a substrate color developing solution A and a substrate color developing solution B; the substrate color development liquid A is at least one of sodium acetate, citric acid and hydrogen peroxide; the substrate color development liquid B is at least one of disodium ethylene diamine tetraacetate, citric acid, glycerol and 3,3',5,5' -tetramethyl benzidine.

Technical Field

The invention relates to the field of molecular biology, in particular to a method and a kit for detecting RNA methylation m 6A.

Background

N6-Methylalodesine (m6A), a modification of adenine nucleotides catalyzed by adenylate methyltransferase, is widely distributed in different types of RNA. It is well known that RNA methylation plays an important role in the variable cleavage, transcription initiation and translation of RNA. The RNA methylation level is closely related to biological processes such as metabolism, genetic development, disease occurrence and development and the like.

Various methods have been developed to efficiently map m6A throughout the transcriptome. The initial study used immunoprecipitation, which utilized a highly specific m6A antibody for next generation sequencing. These methods are called MeRIP-seq. MeRIP has been improved and is now available for single nucleotide resolution m6A localization. This procedure can be achieved by a very small RNA fragment (100- & ltSUB & gt 200nt) combined with an immunoprecipitation reaction, the improved method being called miCLIP and described first by Linder et al 2015. miCLIP has now been applied to the localization of m6A in many different cell types and organisms, revealing its new potential functionality.

There have been technical difficulties in detecting m6A at the single base resolution level. In addition, the low abundance of mRNA/lncRNA carrying m6A, the reaction inertness of the m6A methyl group, and the interference of RNA structure near m6A make it more difficult to detect m6A at the single base level. Currently, the widely applied m6A/MeRIP-seq technology utilizes m6A antibody to carry out co-immunoprecipitation on RNA fragments carrying m6A modification, then RNA sequencing is carried out, and m6A is positioned within the range of 200 nt. Although these techniques make it possible to analyze the apparent transcript profile of m6A, they do not allow precise localization of the adenylate actually modified by m6A in MeRIP-Seq peak, nor do they allow quantification of each modification site. Therefore, in order to further understand the biological functions and molecular mechanisms of m6A epigenetics, a new technology is urgently needed, and the modification state and modification ratio of m6A can be determined at a single base level.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an efficient, sensitive and quantitative RNA methylation m6A detection method and a kit.

In order to achieve the purpose, the invention adopts the technical scheme that:

an RNA methylation m6A detection method, comprising the following steps:

(1) adding the RNA sample into the experiment hole coated by the antibody, and incubating at room temperature;

(2) discarding liquid in the experimental hole, adding washing liquid, blowing and beating by using a liquid transfer gun, discarding the washing liquid, and repeating for three times;

(3) adding a reverse transcription primer into an experimental hole to perform reverse transcription reaction;

(4) adding streptavidin with a label into the experimental hole, and carrying out a binding reaction between the streptavidin and biotin;

(5) discarding liquid in the experimental hole, adding washing liquid, blowing and beating by using a liquid transfer gun, discarding the washing liquid, and repeating for three times;

(6) adding a color developing agent into the experimental hole, incubating, and adding a stop solution;

(7) the absorbance of the mixture in the test well was measured and calculated to give the m6A content.

The content of the target gene m6A can be detected by utilizing the process of synthesizing DNA by taking RNA as a template in the reverse transcription, coupling biotin to a reverse transcription random primer, combining the random primer with an RNA fragment immunoprecipitated by m6A, combining streptavidin with the biotin and further utilizing a TMB color development method.

Preferably, the antibody is an m6A recombinant antibody.

Preferably, the reverse transcription primer is an oligomeric thymine primer, a random primer or a gene-specific primer.

The reverse transcription Random primer can be selected from among an Oligo thymine primer (Oligo dT), a Random primer (Random hexamers) and a Gene Specific Primer (GSP) according to the purpose of the experiment.

If the template is of eukaryotic origin, Oligo dT is generally preferred, paired with the 3' PolyA tail of eukaryotic mRNA, and suitable for reverse transcription of long-chain or even full-length mRNA. It has high quality requirements for RNA samples, and even a small amount of degradation can greatly reduce the synthesis amount of full-length cDNA.

When random primers are selected, all RNAs are used as templates in the first strand cDNA synthesis reaction, and primers are designed for specific amplification when PCR reaction is carried out. Also, taking note of the relationship between the amount of random primers and the amount of total RNA, it is generally recommended that the amount of random primers be 50ng per 5. mu.g of total RNA, and if the amount of random primers exceeds 250ng per 5. mu.g of total RNA, it may result in an increase in small fragment products (<500bp) and a decrease in long fragment and full length products. When the target region has a complex secondary structure or has a high GC content, or the template is of prokaryotic origin, and cDNA synthesis cannot be efficiently primed using Oligo dT or gene-specific primers (GSP), primer Random hexamers can be used.

Preferably, the label is horseradish peroxidase.

Preferably, the incubation temperature is 20-25 ℃, and the incubation time is 1 h.

Preferably, the detection wavelength of the detection absorbance is 450 nm.

An RNA methylation m6A detection kit is characterized by comprising the following materials: washing liquid, sample extracting solution, reverse transcription reagent, negative control liquid, positive control liquid, horseradish peroxidase labeled avidin and stop solution.

Preferably, the washing solution is at least one of sodium chloride, potassium chloride, tris (hydroxymethyl) aminomethane and tween 20.

Preferably, the sample extracting solution is at least one of phenol, guanidinium isothiocyanate and beta-mercaptoethanol.

Preferably, the reverse transcription reagent is at least one of random primer labeled biotin, target gene primer labeled biotin, polythymidine, T repetitive oligonucleotide, deoxyribonucleoside triphosphate, RNase inhibitor and DNA reverse transcription polymerase.

Preferably, the concentration of RNA in the negative control solution without m6A is 100. mu.g/mL.

Preferably, the concentration of m6A oligonucleotide in the positive control solution is 2 μ g/mL.

Preferably, the concentration of the sulfuric acid in the stop solution is 2 mol/L.

2mol/L H can be used after horseradish peroxidase catalyzes TMB to develop blue₂SO₄Terminating the reaction; after addition of sulfuric acid, the solution is yellow, at which time the absorbance can be measured at 450 nm.

Preferably, the RNA methylation m6A detection kit further comprises an enzyme label plate and a horseradish peroxidase-labeled avidin diluent.

Preferably, the horseradish peroxidase-labeled avidin diluent is a balanced salt solution.

The balanced salt solution is a salt solution with the electrolyte content similar to the content in blood plasma.

Preferably, the substrate solution comprises a substrate color developing solution A and a substrate color developing solution B, wherein the substrate color developing solution A is at least one of sodium acetate, citric acid and hydrogen peroxide, and the substrate color developing solution B is at least one of disodium ethylenediamine tetraacetic acid, citric acid, glycerol and 3,3',5,5' -tetramethylbenzidine; mixing solution A and solution B at a ratio of 1:1 before use.

Compared with the prior art, the invention has the beneficial effects that:

(1) the detection cost is low, the synthesis cost of the used reagent is the labeled biotin random primer and the biotin labeled target gene primer is low, and the experimental expense is saved.

(2) The operation is simple and convenient, the operation can be carried out in a basic laboratory only by simple equipment such as a centrifugal machine, a biochemical constant temperature box, an enzyme-labeling instrument and the like, and stable repeatability and reliability are realized.

(3) The detection result can be absolutely quantified.

(4) By adopting the principle of combining the m6A antibody and the detection gene marker primer, the signal specificity can be amplified, the real reliability of a detection sample is improved, and the detection kit has the advantages of high efficiency, high sensitivity and strong specificity.

(5) It is suitable for various samples with free RNA such as serum, plasma, tissue homogenate, cell lysate, etc. If the amount of free RNA in the sample is insufficient, the sample can be extracted, concentrated and detected.

Drawings

FIG. 1 is a schematic diagram of a detection method of m6A RNA methylation.

FIG. 2 is a graph showing the relative quantitative analysis of the RNA samples m6A in examples 1 to 3 in effect example 1.

Fig. 3 is a graph of the standard m6A in effect example 2.

FIG. 4 is a diagram showing an absolute quantitative analysis of the RNA samples m6A of examples 1 to 3 in effect example 2.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific embodiments.