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Single gene mRNA (messenger ribonucleic acid) methylation level detection method

A level detection and methylation technology, applied in the field of molecular biology, can solve the problems of time-consuming and high cost

Inactive Publication Date: 2015-11-04
ZHEJIANG UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently the only method for measuring mRNA methylation is based on the specific recognition of m 6 Next-generation sequencing of immunoprecipitation of antibody A, but this method is costly and time-consuming, so it is very important to establish an inexpensive, simple and easy-to-operate method for the detection of relative levels of methylation in a single gene

Method used

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  • Single gene mRNA (messenger ribonucleic acid) methylation level detection method
  • Single gene mRNA (messenger ribonucleic acid) methylation level detection method
  • Single gene mRNA (messenger ribonucleic acid) methylation level detection method

Examples

Experimental program
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Embodiment 1

[0041] 1. Total RNA extraction:

[0042] Take 50-100mg of two groups of pig liver samples and perform the following operations: after grinding in liquid nitrogen, add 1ml Trizol lysate and mix well, add 0.2ml chloroform and shake vigorously for 15 seconds, incubate at room temperature for 10min, then centrifuge at 4°C and 15000g for 15min , transfer the upper aqueous phase (about 500 μl) to a new tube, add 500 μl of isopropanol, mix upside down; centrifuge at 15,000 g for 10 min at 4°C, discard the supernatant; add 1ml of 75% (volume %) ethanol to wash, 4°C , Centrifuge at 15,000g for 10min, discard the supernatant, and when the RNA precipitate at the bottom of the tube becomes colorless and transparent, add 80μl of DEPC water and blow to mix.

[0043] 2. RNA fragmentation:

[0044] 1) Mix the RNA obtained in step 1 with the fragmentation buffer according to the following reaction system,

[0045] RNA Fragmentation System

[0046]

[0047] In order to obtain the optimal ...

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Abstract

The invention discloses a single gene mRNA (messenger ribonucleic acid) methylation level detection method. The method includes the steps of 1), designing fluorescent quantitative primers in a methylated region of a target gene; 2), extracting total RNA and fragmenting the total RNA into RNA fragments by a chemical fracture method; 3), enabling the RNA fragments obtained in the step 2) to precipitate all mRNA fragments with m6A loci through antibodies of specific recognition m6A, and collecting and enriching by agarose magnetic beads; 4), subjecting the mRNA fragments obtained by enrichment of the antibodies in the step 3) to reverse transcription, and using the fluorescent quantitative PCR (polymerase chain reaction) primers in the step 1) for real-time fluorescent quantitative PCR so as to obtain a relative expression quantity of the methylated target gene, wherein the relative expression quantity represents single gene mRNA methylation level.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a single gene-based mRNA methylation level detection method and primers used. Background technique [0002] Discovered in 1974, RNA methylation modification is the most common post-transcriptional modification, accounting for about two-thirds of all RNA modifications. In eukaryotes, the most common post-transcriptional modification of mRNA is the methylation modification on the 6th N atom of base A, m 6 A accounts for about 0.1%-0.4% of the total adenosine content of cellular mRNA, that is, each mRNA in mammals contains 3-5 6-methyl-modified adenosine on average. m 6 A methylation site mainly occurs in the highly conserved RRACH (R=G,A; H=A,C,T) sequence, which may play an important role in epigenetics, but due to m 6 The A modification method does not destroy the pairing between bases, so it cannot be positioned on the genome by direct sequencing. At the same time...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2600/154C12Q2545/113C12Q2565/113C12Q2563/107
Inventor 汪以真王新霞江芹蔡旻
Owner ZHEJIANG UNIV
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