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Method and kit for detecting RNA N6-methyladenosine modification at single-base resolution in range of whole transcriptome

A technology of methyl adenine and allyl adenine, applied in biological testing, biochemical equipment and methods, analytical materials, etc., can solve problems such as inability to introduce cells

Active Publication Date: 2020-05-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under the current culture conditions, amino acids with modified groups and normal amino acids are at a disadvantage in the competition of cell metabolism and cannot be introduced into cells.

Method used

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  • Method and kit for detecting RNA N6-methyladenosine modification at single-base resolution in range of whole transcriptome
  • Method and kit for detecting RNA N6-methyladenosine modification at single-base resolution in range of whole transcriptome
  • Method and kit for detecting RNA N6-methyladenosine modification at single-base resolution in range of whole transcriptome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 In HeLa cells, m on the transcriptome mRNA 6 A detection of modification sites

[0081] 1. HeLa cell culture, allyl labeling and mRNA extraction

[0082] (1) After the HeLa cells were cultured to 80% fullness under normal culture conditions, the medium was aspirated, and the residual medium was washed with phosphate-buffered saline (PBS);

[0083] (2) 10% fetal bovine serum (FBS), 1% 100x penicillin-streptomycin double antibody, and 1 mM cysteine ​​were added to the medium not containing methionine, and the above-mentioned HeLa cells were Incubate in this medium for 30 minutes to remove residual methionine in the cells;

[0084] (3) After removing methionine, add 1 mM allyl-L-seleno / thiohomocysteine ​​to the medium for culturing the above HeLa cells, and continue culturing for 16 hours;

[0085] (4) After the cells have been cultured, suck off the medium, wash off the residual medium with PBS, and then add TRIzol to the culture dish TM Cover the bottom of t...

Embodiment 2

[0164] Example 2 In mouse H2.35 cells, m 6 A detection of modification sites

[0165] 1. Mouse H2.35 cell culture, allyl labeling and mRNA extraction

[0166] (1) Mouse H2.35 cells were cultured under normal culture conditions (with the addition of 200nM DEX, dexamethasone), cultured to 60% of the fullness, sucked off the medium, and washed with phosphate-buffered saline (PBS) Remove residual medium;

[0167] (2) Add 10% fetal bovine serum (FBS), 1% 100x penicillin-streptomycin double antibody, 200nM DEX (dexamethasone), and 1mM cysteine ​​to the medium without methionine , culturing the above-mentioned mouse H2.35 cells in this medium for 30 minutes to remove residual methionine in the cells;

[0168] (3) After removing methionine, add 1 mM allyl-L-seleno / thiohomocysteine ​​to the medium for culturing the above-mentioned mouse H2.35 cells, and continue culturing for 16 hours;

[0169] (4)-(12) The culture, allyl labeling and mRNA extraction of HeLa cells are the same as t...

Embodiment 3

[0175] Example 3 Culture of HeLa cells and mouse H2.35 cells under normal cell culture conditions

[0176] 1. Culture of HeLa cells

[0177] Add 10% fetal bovine serum (FBS) and 1% 100x penicillin-streptomycin double antibody to normal cell culture medium, culture HeLa cells in this medium for 16-24 hours, then extract HeLa cells mRNA, its m 6 A content such as figure 2 Shown in Ctrl.

[0178] At the same time, HeLa cells were cultured according to the treatment conditions of the above-mentioned allyl labeling method, and 1 mM allyl-L-seleno, allyl-L-thiohomocysteine, or methionine (methionine) was added. Base-L-thiohomocysteine), cultivated for 16 hours, to obtain the m of mRNA 6 A content such as figure 2 In Se-allyl-L-selenohomocysteine, S-allyl-L-homocysteine, L-Methionine shown. figure 2 Se-allyl-L-selenohomocysteine / S-allyl-L-homocysteine ​​is allyl-L-selenohomocysteine / thiohomocysteine ​​introduced into HeLa cells to obtain N in mRNA 6 -The content of allyl ad...

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Abstract

The invention provides a method and kit for detecting RNA N6-methyladenosine modification at single-base resolution in a range of a whole transcriptome. According to the method, on the basis of an N6-allyl label of in-vivo ribonucleic acid (RNA) adenine and chemical treatment, base mutation of the in-vivo ribonucleic acid (RNA) adenine in a process of reverse transcription into DNA is induced, andthen a mutation site is recognized by means of nucleic acid sequencing, so that an a6A site is obtained, and the a6A site is a site originally modified by m6A in cell RNA. By means of the method, thespecific label of N6-allyladenine in a cell is achieved for the first time, and the label not only can be used for replacing an N6-methyladenosine site in the cell, but also can be positioned by means of mutation sequencing. Compared with existing gene sequencing technologies applied to m6A detection, the method for detecting RNA N6-methyladenosine modification at single-base resolution in the range of the whole transcriptome has the advantage that due to the fact that the mutation site can be accurate down to single-base resolution, the precision of m6A site detection based on m6A antibody immunoprecipitation and a massively parallel sequencing method which are currently and generally adopted is improved, so that the method for detecting RNA N6-methyladenosine modification at single-baseresolution in the range of the whole transcriptome is a direct high-throughput single-base identification method.

Description

technical field [0001] The invention belongs to the field of gene sequencing, in particular to a whole-transcriptome-wide single-base resolution detection RNA N 6 -Methods and kits for methyladenine modification. Background technique [0002] RNA is not only composed of four bases, cytosine (C), thymine (U), guanine (G), and adenine (A). N 6 -Methyladenine (m 6 A) is an extremely important modified base on RNA, which plays a variety of biological functions in biological processes, such as regulating gene expression and so on. Among them, the identification and sequencing methods of modifications are the prerequisites for studying their biological significance. [0003] Since the physicochemical properties of methyl-modified adenine are close to those of ordinary adenine, it cannot be directly detected by the existing first-generation or next-generation sequencing technology. Currently, based on m 6 A antibody immunoprecipitation sequencing technology (MeRIP-seq) allows...

Claims

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Application Information

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IPC IPC(8): C12Q1/6809C12Q1/6869G01N33/53
CPCC12Q1/6809C12Q1/6869G01N33/5308C12Q2521/107C12Q2525/117C12Q2525/191C12Q2535/122
Inventor 刘建钊冯新华舒潇曹婕
Owner ZHEJIANG UNIV
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