CN111434778A - RNA molecule m6A modification level detection method and application - Google Patents
RNA molecule m6A modification level detection method and application Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The present invention relates to a method for detecting the level of modification of RNA m6A in a biological or medical sample. The method is characterized in that the method is based on the characteristic that YTHDF2 protein is combined with RNA molecule m6A to interfere reverse transcription reaction of reverse transcriptase on the RNA molecule, and then the specific RNA molecule modified at a specific site m6A is detected by a PCR amplification technology. The method can be used for detection and analysis of m6A modified RNA molecules in biological or medical samples.
Description
The invention belongs to the technical field of biomedicine, and particularly relates to a detection method of a m6A modified RNA molecule, which can be used for detection analysis of the m6A modified RNA molecule and detection of biological and clinical specimens.
Background RNA base modifications are ubiquitous in RNA molecules. Recent studies have shown that different base modifications in RNA molecules have important biological functions, and that dynamic Modification changes of bases in RNA molecules are related to the development of various diseases (Batista PJ. the RNA Modification N (6) -Methaladenosine and Its indications in human diseases. genomics biology information.2017, 15 (3): 154-. Therefore, there is an increasing interest in the base modification studies of RNA molecules. The most studied and clearly understood is the m6A modification of RNA molecules. However, the current analysis method for the modification of RNA molecule m6A mainly comprises obtaining data by a coprecipitation high-throughput sequencing (meRIP-seq) method based on the RNA methylation (m6A) of m6A antibody, and then analyzing by bioinformatics method to obtain the information of m6A modification of RNA molecule (dominisini D, et al. polarity of the human and genome m6A RNA methylation modified by m6A-seq. Nature.2012, (485) (7397): 201): 206; Chen K, et al. high-resolution N6-methyl thionine (m6A) map using phosphor to cross linked-identified m (6) A sequencing. Angel M Int Engl. 54, 1585) 1590). Although a series of different methods have been derived based on the above-described method, such methods are more suitable for analyzing the overall modification status of RNA molecules. Currently, methods for detecting and analyzing RNA molecules modified at specific sites are under development. Goovina et al established a dissolution curve change detection method (Goovina AY, et al. method for site-specific detection of m6A nuclear expression in RNA based on high-resolution (HRM) analysis. nucleic Acids Res.2014, 42 (4): e27), Hong et al established an interference reverse transcription detection method (Hong T, et al. precursor Antibody-induced m6A Identification of 4 dTTP and FTO-Assisted expression) based on participation of 4SedTTP and FTO (surface and affinity) and a method for detecting Single-Nucleotide-reaction solution. jam chemistry, sample chemistry, 2018: 5889). These methods can detect m6A modified RNA at a specific site. These methods have their own features, but they also have their disadvantages.
Detection of changes in the level of modification of m6A at specific sites in specific RNA molecules has important implications for biological functional studies of m6A modified RNA, and for detection of modification of m6A at specific sites in specific RNA molecules in disease-related clinical samples, which makes it impossible to monitor and analyze specific RNA molecules modified at specific sites m6A in conventional detection and analysis methods simply, rapidly and on a large scale because of high similarity of m6A to A and in most cases low proportion of modification of m6A in RNA molecules, recent studies have found that various protein molecules are involved in the modification of m A of RNA molecules, including modified enzymes, removed enzymes and related binding protein molecules, among which YTHDF2 (H-binding protein 2) has the function of binding with m6A naturally and specifically (ZHU T, patent, CRYSTALS T of the modification of the same family of the same gene protein gene 2, 1496 binding protein molecules 1496, 1496-1496 binding protein molecules found in 1499-14 protein molecules 1496, 1496-1496 binding protein molecules found in the detection method (see No. 7. 12).
The invention aims to establish a simple method for detecting m6A modified RNA molecules, realize the detection of the RNA molecules modified at a specific site m6A, and be suitable for the detection and analysis of biological samples and clinical samples. The detection method is characterized in that YTHDF2 protein is added into a reverse transcription reaction system for RN A by utilizing the characteristic that YTHDF2 protein binds to m6A methylation modification sites in RNA molecules, YTHDF2 protein molecules bind to sites with m6A modification in RNA, and then the extension of reverse transcription primers in the RNA reverse transcription reaction is hindered, and a cDNA short-fragment reverse transcription product which is terminated before the m6A modification sites is obtained. Through designing a pair of PCR reaction primers spanning m6A modification sites and a pair of PCR amplification primers at the 3' side of the m6A modification sites of the RNA molecules, the reverse transcription products are respectively amplified by using the two pairs of primers, and through comparing the amplification results, the detection and analysis of the m6A modification RNA molecules are realized.
The detection method of the m6A modified RNA molecule comprises two main steps: (1) and (5) reverse transcription reaction. One group of normal reverse transcription reactions, and one group of reverse transcription reactions in which YTHDF2 protein is added to bind with RNA molecules. (2) And (3) carrying out PCR reaction. PCR reactions were performed using PCR primers spanning the m6A modification site and PCR amplification primers 3' to the m6A modification site of the RNA molecule, respectively. According to different modes of PCR reaction detection amplification products, common PCR reaction can be adopted, and fluorescent dye or molecular beacon can be added to adopt real-time quantitative PCR reaction. Depending on the purpose of detecting RNA molecules, PCR detection can also be performed by using only reverse transcription reaction products which are combined with RNA molecules by adding YTHDF2 protein.
In the method of the invention, the YTHDF2 protein used can be separated and purified from human cell lines and tissues, and can also be a fusion protein expressed in prokaryotic and eukaryotic expression systems. In this example, the use of separation and purification in Escherichia coli expression in His tag recombinant human YTHDF2 protein. In a specific reaction, the using amount of the YTHDF2 protein is determined according to the using amount of RNA in a reverse transcription reaction and the modification degree of m6A in the RNA, so that the blocking effect meeting the detection requirement is achieved. In the commonly used reverse transcription system, the amount of YTHDF2 protein may be several micrograms to several hundred micrograms.
In the reverse transcription reaction, the amount of reverse transcriptase and the amount of dNTPs have an influence on the efficiency of the reverse transcription reaction. In general, within a certain range, the reverse transcription reaction proceeds favorably by increasing the amount of reverse transcriptase used and increasing the concentration of dNTP. The method of the invention uses the characteristic that YTHDF2 binds to the m6A modification site in the RNA molecule to block the reverse transcription reaction. In order to achieve the blocking effect required for detection, the amount of reverse transcriptase may be the amount recommended for a reverse transcription kit generally used, or may be reduced to one eighth or less of the recommended amount. The concentration of dNTPs may be the amount recommended for the use of a reverse transcription kit, or may be reduced to 1. mu.M or less. The blocking effect meeting the detection requirement is achieved according to the amount of the RNA used in the reverse transcription reaction and the modification degree of m6A of the RNA molecule.
The invention provides a method for detecting methylation of RNA m6A, which comprises determining one or more RNAs in a sample. In the actual detection process, any one RNA molecule can be detected, and m6A methylation of different RNA molecules can also be detected, so that the change of the methylation level of the RNA molecule m6A in the sample can be determined.
The RNA m6A detection method is suitable for body fluid sample detection, including urine, saliva, blood derivatives and the like, and cell and tissue samples and the like. The preparation of RNA in different samples adopts a conventional RNA preparation method.
For convenient detection, the kit can be assembled according to the method and used, and the kit consists of a conventional reverse transcription reagent, a PCR or real-time quantitative PCR reagent and YTHDF2 protein.
The invention is illustrated by the following examples, which are intended to be illustrative only and not to be limiting in any way. The specific experimental methods not mentioned in the examples are generally carried out according to the conventional experimental methods.
The specific implementation method of the invention is described with reference to the attached drawings.
Drawings
FIG. 1: schematic principle diagram of m6A modified RNA molecule detection method
FIG. 2: detection of synthetic m6A modified RNA molecules
FIG. 3: detection of m6A modified 18S rRNA and 28S rRNA
Examples
Example one
Detection of synthetic m6A modified RNA molecules
1. Synthesis of m6A modified RNA molecule, reverse transcription primer and PCR reaction primer
The synthesized RNA fragment contained the m6A modification at position 40.
m6A modified RNA molecule (MeA): 5' -UCGGCCCACGGCCCUGGCGGAGCGCUGAGAAGACGGUCGA (m6A) CUUGACUAUCUAGAGGAAG.
No m6A modified RNA molecule (MeU): 5' -UCGGCCCACGGCCCUGGCGGAGCGCUGAGAAGACGGUCGACUUGACUAUCUAGAGGAAG.
Reverse transcription primer (MeA-U-RT primer): 5' -CTTCCTCTAGATAGTCAAGTTC.
PCR Forward primer (Forward primer): 5' -TCGGCCCACGGCCCTGGCGG.
PCR reverse primer (Reversed primer): 5' -CTTCCTCTAGATAGTCAAGTTC.
2. Reverse transcription reaction
With GoScriptTMTaking a mixture of MeA and MeU as a Reverse transcription template, taking the mixture of MeA and MeU in different reaction systems as 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10% respectively, taking 100ng of RNA to be detected as the Reverse transcription template, respectively adding 1 mu L (0.5 mu g) Reverse transcription primer (MeA-U-RT ptimer), supplementing DEPC water to 5 mu L, placing in a metal bath at 70 ℃ for 5min, then placing on ice for 5min, respectively adding 4 mu g YTHDF2 protein (recombinant His-YTHDF2 fusion protein expressed by escherichia coli used in the experiment) to incubate at 37 ℃ for 20min, centrifuging at 3000r/min for 20s, and then adding 4 mu l 5 × RT buffer solution, 1 mu L dNTP (0.1mM), and 2.4 mu l Mgcl to each tube2(25mM), 0.5. mu.l (20U) of RNase inhibitor, 0.5. mu.l (100U) of GoScriptTM reverse transcriptase, and made up to 20. mu.l with RNase-free water. In a metal bath at 25 ℃ for 5min and at 42 ℃ for 60min,the cDNA obtained is stored at-20 ℃ for 15min at 70 ℃ or directly used in PCR reaction.
PCR reaction and agarose gel electrophoresis analysis
The real-time quantitative PCR reaction system comprises 1 mu L cDNA template, 10 mu L2 × TaqPCR starMix, 0.5 mu L (20mM) PCR forward primer, 0.5 mu L (20mM) PCR reverse primer and water to supplement 20 mu L, the reaction conditions are pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, denaturation at 60 ℃ for 30s and amplification at 72 ℃ for 30 cycles, and the PCR reaction product is subjected to agarose gel electrophoresis for result analysis.
The results of the tests are shown in FIG. 2, and FIG. 2 shows the results of 3 replicates. Reverse transcription of the m6A modified synthetic RNA fragments was prevented using 4. mu.g YTHDF2 protein in this experiment.
Example two
RNA molecule for detecting m6A modified 28S rRNA
1. Reverse transcription primer and PCR primer design
Synthetic reverse transcription primers and PCR primers were designed for detecting m6A modification at position 4190 of 28S rRNA.
28S rRNA reverse transcription primer (28S-RT): 5' -AATCAAGATCAAGCGAGCTT. This primer is also the downstream primer of the PCR amplification reaction.
28S rRNA forward primer 1 (28S-Fu): 5' -GGCGCGACCCGCTCCGGGGA. This primer is located 5' to the m6A modification site.
28S rRNA forward primer 2 (28S-Fd): 5' -CGCAGGTGTCCTAAGGCGAG. This primer is located 3' to the m6A modification site.
2. Reverse transcription reaction, PCR reaction and agarose gel electrophoresis conditions were the same as in example one
In this experiment 2. mu.g YTHDF2 protein was used. The detection result is shown in FIG. 3, and the detection of m6A modified RNA is realized. The results were calculated as follows: Δ Ct0 ═ Ct0(Fu) -Ct0(Fd), Δ Ctx ═ Ctx (Fu) -Ctx (Fd),
the Rx/R0 ratio was compared.
Claims (8)
1. An assay for detecting a modified RNA molecule of m6A, comprising: based on the total RNA in purified cells, tissues and body fluids, m6A binding protein YTHDF2 is added in the reverse transcription reaction of the total RNA, so that the total RNA is bound with m6A in RNA molecules to interfere the progress of the reverse transcription reaction, a cDNA fragment which is terminated before a m6A modification site is obtained, a pair of primers spanning the m6A modification site and a pair of primers at one side of the m6A modification site are used for respectively carrying out PCR reaction on the cDNA obtained by reverse transcription, and the Ct values of the two groups of PCR reactions are compared to achieve the aim of detecting the m6A modified RNA.
2. The detection method according to claim 1, wherein: comprises two main steps of (1) reverse transcription reaction; (2) and (3) carrying out PCR reaction.
3. The m 6A-binding protein YTHDF2 used in reverse transcription reaction according to claim 1, wherein: YTHDF2 protein is purified YTHDF2 protein, or recombinant YTHDF2 protein or YTHDF2 fusion protein, and the dosage of YTHDF2 protein in reaction is 1-100 micrograms.
4. The reverse transcription reaction according to claim 1, wherein: the dosage of the reverse transcriptase is from the conventional dosage of the reverse transcription reaction to one eighth of the conventional dosage; the amount of dNTPs used is from the conventional amount for a reverse transcription reaction to one percent of the conventional amount.
5. The primers for PCR reaction as set forth in claim 1, wherein: wherein, the upstream primer and the downstream primer in one pair of primers are respectively positioned at two sides of the modification site of the RNA molecule m6A, and the upstream primer and the downstream primer in the other pair of primers are respectively positioned at one side of the 3' end of the modification site of the RNA molecule m 6A.
6. The PCR reaction of claim 1, wherein: and adding fluorescent dye or molecular beacon into a reaction system of the PCR reaction to perform real-time quantitative PCR reaction.
7. Use of the detection method of any one of claims 1 to 6 in a m6A modified RNA molecule detection assay.
8. The method for detecting an RNA molecule of any of claims 1 to 6, wherein the sample comprises a cell, urine, saliva, blood derivative or tissue sample.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102317473A (en) * | 2008-12-11 | 2012-01-11 | 加利福尼亚太平洋生物科学股份有限公司 | Shenzhen tcl new technology co. , ltd |
| CN105018617A (en) * | 2015-07-20 | 2015-11-04 | 浙江大学 | Single gene mRNA (messenger ribonucleic acid) methylation level detection method |
| US20180245128A1 (en) * | 2015-08-31 | 2018-08-30 | The University Of Chicago | Composition and methods for detecting adenosine modifications |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102317473A (en) * | 2008-12-11 | 2012-01-11 | 加利福尼亚太平洋生物科学股份有限公司 | Shenzhen tcl new technology co. , ltd |
| CN105018617A (en) * | 2015-07-20 | 2015-11-04 | 浙江大学 | Single gene mRNA (messenger ribonucleic acid) methylation level detection method |
| US20180245128A1 (en) * | 2015-08-31 | 2018-08-30 | The University Of Chicago | Composition and methods for detecting adenosine modifications |
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| Title |
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| YU XIAO 等: "An Elongation- and Ligation-Based qPCR Amplification Method for the Radiolabeling-Free Detection of Locus-Specific N6-Methyl-adenosine Modificatio", 《ANGEW. CHEM. INT. ED.》 * |
| 苏晨 等: "m6A修饰RNA结合蛋白YTHDF2 的克隆、表达及其活性分析", 《军事医学》 * |
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