Method for detecting RNA m6A with high flux, high sensitivity and single base resolution and application thereof

Abstract

The invention discloses a method for detecting RNA m6A with high flux, high sensitivity and single base resolution and an application thereof. The method comprises the following steps: (1) extractingtotal RNA in a sample and removing rRNA to obtain an RNA sample; (2) fragmenting the RNA sample, and dividing the fragmented RNA sample into two parts, namely an RNA fragment I and an RNA fragment II;(3) carrying out co-immunoprecipitation and cross-linking treatment on the RNA fragment I; (4) respectively carrying out 3'tail end joint connection on the complex obtained by cross-linking treatmentand the RNA fragment II; (5) respectively carrying out reverse transcription to obtain cDNA, connecting the cDNA with a 3'end joint, carrying out PCR amplification, and respectively constructing an hmCLIP library and an input library; and (6) sequencing the hmCLIP library and the input library by using a high-throughput sequencing platform. The method is short in consumed time, high in sensitivity and suitable for detecting RNA m6A in a small number of specimens and specimens with low RNA integrity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for detecting RNAm6A with high-throughput and high-sensitivity single-base resolution and its application. Background technique [0002] The m6A modification of RNA occurs at the N6 position of adenine nucleotide in RNA, which is the most common type of apparent post-transcriptional modification on eukaryotic mRNA, accounting for about 0.1-0.4% of adenine on mRNA. The m6A modification process of RNA is catalyzed by the methyltransferase complex and demethylated by the demethyltransferase, so it is a dynamically reversible process. More and more studies have shown that m6A plays an important role in important life processes such as embryonic development, neurogenesis, tumor progression, and sex determination. Therefore, it is extremely important to accurately locate the m6A modification site of RNA to study the specific mechanism of m6A regulating life activities. However, ...

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Problems solved by technology

Although both meRIP and miCLIP can detect m6A information at the whole transcriptome level, there are still some shortcomings: 1. These two methods require a large amount of sample RNA, and the general amount of RNA is about 500ug. It is difficult to realize the detection of m6A in the medium; second, meRIP is realized by fragmenting the RNA and enriching it according to the m6A antibody, and the final result is a fragment with m6A sites enriched by the antibody, and the resolution is low; 3. miCLIP only determines the mutation site through the peaks obtained by antibody enrichment, and there will be a high false positive rate for RNA with a high background expression level; 4. The process of miCLIP is cumbersome, with many steps, and requirements for experimental conditions higher

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