Method for high-flux sequencing for establishing of human miRNA (micro-ribonucleic acid) sequencing library

Abstract

The invention discloses a method for high-flux sequencing for establishing of a human miRNA (micro-ribonucleic acid) sequencing library. The method comprises the following steps of S1, extracting human total RNA, and reversing into cDNA (complementary deoxyribonucleic acid); S2, using the cDNA as a template, using a probe to perform mmPCR (multi-multiplex polymerase chain reaction) pre-amplification and mmPCR amplification on miRNA in a sample, and respectively connecting a 3' terminal and a 5' terminal to both ends of an amplified product; S4, connecting a tag sequence with a mmPCR amplifiedproduct; S5, performing gel electrophoresis on the sample added with the tag sequence, recycling a pure electrophoresis product, and building the miRNA library; S6, mixing the obtained multiple miRNAlibraries, and utilizing a next generation sequencing technique to perform high-flux sequencing. The method has the advantages that the detection sensitivity of RNA editing and modifying on the precursor miRNA is greatly improved, the uniformity of the RNA-targeted sequencing libraries is improved, the library building and sequencing cycle is shortened, the cost is reduced, and the application prospect is larger.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a method for constructing a human miRNA sequencing library for high-throughput sequencing. Background technique [0002] Since 2006, nucleic acid sequencing technology has made a revolutionary breakthrough. Next Generation Sequencing technology (NextGeneration Sequencing, NGS) can quickly determine millions of sequences, technically realizing a comprehensive analysis of the transcriptome and genome of a species . In particular, the generation of high-throughput transcriptome sequencing technology (RNA-seq). Transcriptome sequencing (RNA-seq) is the use of NGS technology to sequence, comprehensively and quickly obtain almost all transcripts of a species or a specific organ or tissue in a certain state, and analyze the entire transcriptome, tissue or cell in an individual, tissue or cell. Small RNA content, or gene expression profiling, is currently a p...

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Problems solved by technology

[0004] However, the current mmPCR-seq is mainly suitable for the effective construction of mRNA library sequencing, and the construction effect of miRNA library is not very good.

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