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M6A RNA methylation site detection method

A detection method, methylation technology, applied in the field of molecular biology, can solve the problems that hinder the understanding of human diseases and restrict research, and achieve the effects of reducing errors, high RNA integrity, and simple experimental procedures

Active Publication Date: 2022-02-01
北京恩泽康泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional m6A-seq detection in the existing literature and reports requires hundreds of micrograms of RNA, which severely limits the research on many clinical micro-samples and hinders the understanding of human diseases, especially tumors, cancers and other diseases

Method used

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  • M6A RNA methylation site detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Extraction of Trace Cell Total RNA

[0072] The present invention provides a method for detecting m6A methylation in a trace amount of total cellular RNA (see figure 1 ), providing a method for detecting m6A methylation sites in mRNA and IncRNA and its application.

[0073] The present invention adopts the provided ultra-trace rRNA strand-specific m6A methylation detection library construction kit, and the method includes but is not limited to one or more of the following steps:

[0074] 1. Sample pretreatment: the sample used in this example is 293-T cells. Firstly, the cultivated 10 6 Collect 293-T cells, centrifuge at 500×g for 5-8min at room temperature, discard the supernatant and leave the cell pellet.

[0075] 2. Genome-free total RNA extraction: Extract total RNA from 293-T cell samples using the column extraction method (QIAGEN, Cat. No. 217204). During the extraction process, pay attention to using it in advance to eliminate the genome in the tota...

Embodiment 2

[0084] Example 2 Capture of m6A RNA fragments

[0085] 1. Prepare antibody magnetic bead RNA mixture: take 10µg of total RNA as an example, prepare according to the following system, mix well by inverting up and down, and incubate at room temperature for 1.5 hours on a rotator;

[0086]

[0087] RNA sample concentration is 1µg / µL, m6A-specific antibody 2µg / tube;

[0088] 2. Lysis of total RNA: After the incubation, add 10 µL RNase inhibitor and 2 µL nuclease lyase to the mixture in the previous step, and lyse at room temperature for 2-4 minutes;

[0089] 3. Enrichment of m6A methylated RNA: after lysis, place it on a small magnetic stand for 2-3 minutes, then remove the supernatant by magnetic separation;

[0090] 4. Elution of non-m6A RNA fragments: Add 150 µL of low-salt buffer and high-salt buffer to wash 3-4 times;

[0091] 5. Purification of m6A RNA fragments:

[0092] 1) Prepare 20µL proteinase K solution / tube from the purified complex in low-salt buffer and high-s...

Embodiment 3

[0096] Embodiment 3 m6A RNA library construction

[0097]The RNA products enriched in Example 2 were subjected to rRNA strand-specific library construction.

[0098] 1. Configure one-strand cDNA synthesis: configure on ice according to the following system;

[0099]

[0100] 1) Put the above mixture on ice immediately after running the following reaction procedure: 72°C, 3 minutes, 2 minutes on ice;

[0101] 2) After preparing the mixed solution according to the following system, take the Mix and add it to the reaction solution in the previous step:

[0102]

[0103] 3) After mixing by pipetting, run the following program: 90min at 42°C; 10min at 70°C; ∞ at 4°C.

[0104] 2. Prepare PCR1 reaction

[0105] 1) Configure the reaction mix according to the following reagents, mix by pipetting and add to the reaction solution in step 1:

[0106]

[0107] 2) Run the following program after brief centrifugation by pipetting and mixing: 94°C for 1min; 98°C for 15sec, 55°C f...

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Abstract

The invention provides an m6A RNA methylation site detection method, and belongs to the field of molecular biology. The method is specially used for detecting the m6A site aiming at the low-initial-amount total RNA, the whole experiment process is simple, RNA integrity is high, library quality is good, the method is especially suitable for the situation that the initial amount of a clinical sample is small, efficient m6A methylated RNA obtaining can be achieved, the library with the high enough concentration is finally obtained through rRNA chain removal specific library building, the application related to computer sequencing and subsequent sequencing is completed, identification of m6A methylation sites in mRNA and IncRNA of clinical trace samples and precious clinical samples is effectively achieved, and the method can be applied to m6A methylation research in the fields of mechanism discussion, diagnosis and treatment and the like.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting m6A RNA methylation sites. Background technique [0002] RNA modification is one of the important contents of epigenetics, and it is a post-transcriptional regulation method. More than 170 kinds of RNA modifications have been identified, which widely exist in various types of RNA, such as mRNA, tRNA, rRNA, sncRNA and IncRNA. The most common RNA post-transcriptional modification methods are N6-methyladenosine (N6-methyladenosine, referred to as m6A, that is, methylation on the sixth nitrogen atom of RNA bases) and 5-methylcytidine (C5-methylcytidine, Abbreviated as m5C, that is, there is an RNA methylation at the 5th position of the cytidine base, etc.). [0003] RNA methylation modification accounts for more than 60% of all RNA modifications, and m6A (6-methyladenine) is the most abundant form of RNA methylation modification on mRNA in higher organisms (m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2527/127C12Q2521/107C12Q2521/327C12Q2521/537
Inventor 程敏秦伟伟林海军陈静李志孔关义
Owner 北京恩泽康泰生物科技有限公司