Method for increasing the yield of m<6>A antibody-enriched methylation mRNA

A methylation and antibody technology, applied in the field of medical biology, can solve the problems of shortening the test time, high price, and reducing the test cost, and achieve the effect of saving the test time, reducing the cost, and improving the accuracy.

Inactive Publication Date: 2017-11-03
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to m 6 A problem of low yield and expensive co-immunoprecipitation of antibody and mRNA, providing a method for eluting m 6 A and the eluent of the methylated mRNA complex and its application, a

Method used

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  • Method for increasing the yield of m&lt;6&gt;A antibody-enriched methylation mRNA
  • Method for increasing the yield of m&lt;6&gt;A antibody-enriched methylation mRNA
  • Method for increasing the yield of m&lt;6&gt;A antibody-enriched methylation mRNA

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Embodiment 1

[0023] 1. Cell culture

[0024] HepG2 cells were treated with 1×10 6 Planted in a 10cm Petri dish in CO 2 The concentration is 5%, the temperature is 37° C., and the humidity is 95% in a carbon dioxide incubator for 48 hours.

[0025] 2. Extraction of total RNA

[0026] After the cells were cultured for 48 hours, the medium was removed, washed once with PBS, 3ml of TRIzol was added, the cells were blown off with a pipette gun, and the cell suspension containing TRIzol was dispensed into 1.5ml centrifuge tubes. Add 200 μl of chloroform to each ml of TRIzol suspension, place it on a vortex shaker for 5 seconds, and then let it stand for 2-3 minutes. After the liquid was separated, it was centrifuged at 12000 rpm for 15 min at 4°C. After centrifugation, the liquid is divided into 3 layers, and carefully pipette about 500 μl of the uppermost liquid into a new 1.5ml centrifuge tube. Add an equal volume of isopropanol, shake and mix, and let stand at room temperature for 10 min...

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Abstract

The invention provides a method for increasing the yield of m<6>A antibody-enriched methylation mRNA. The method is characterized in that a prepared compound of m<6>A and methylation mRNA is eluted by eluent to obtain methylation modified mRNA, and the methylation modified mRNA is used for target gene methylation degree qRT-PCR analysis or high-throughput sequencing to analyze the methylation features of genes; the eluent comprises 0-0.2M of NaCl, 1-20mM of Tris with pH being 8.0, 0.5-5mM of EDTA, 0.01-0.5 of SDS and 1-300 microgram/L of proteinase k. The method has the advantages that the method is different from other methods, the method uses the protease k to elute the compound of the m<6>A and the mRNA, the methylation mRNA high in abundance is obtained, test cost is lowered greatly, test time is shortened, and an economical and effective test method is provided for the researches of m<6>A methylation modification.

Description

Technical field: [0001] The invention belongs to the field of medical biotechnology, in particular to a method for improving m 6 A method for antibody enrichment of methylated mRNA yield. technical background: [0002] Messenger RNA (mRNA) is the core molecule connecting DNA and protein, and the process of genetic information transmission it participates in is the core process of life. Although the modification of mRNA has been discovered in the 1970s, due to the characteristics of mRNA itself and the limitation of technology, the research progress of mRNA modification is relatively slow. with m 6 A-seq, MeRIP, miCLIP, m 1 A-seq, m 1 A series of technologies such as A-ID-seq, Aza-IP, hMeRIP-seq, ψ-seq, CeU-seq, and RiboMeth-seq generate transcriptome-wide m 6 A. m 1 A. m 5 Modification site information such as C, 5hmC, pseudouracil modification and 2'-O-methylation were revealed. In the past two years, there has been a blowout development in mRNA modification researc...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1003C12Q1/6806
Inventor 钟翔余嘉瑶李兴美李毅王恬张莉莉王超
Owner NANJING AGRICULTURAL UNIVERSITY
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