Transcriptome sequencing method based on RT-WES (reverse transcription-whole exome sequencing) technology

Abstract

The invention discloses a transcriptome sequencing method based on RT-WES (reverse transcription-whole exome sequencing) technology. The method comprises the following steps: 1) extracting total RNA of a sample; 2) synthesizing cDNA with total RNA as template; 3) constructing a cDNA fragment library; 4) capturing whole exons of the fragment library and enriching transcriptome fragments of functional genes; 5) sequencing the transcriptome fragments and analyzing sequencing data. The RT-WES technology is adopted, RNA is first converted into DNA through cDNA synthesis process, and then exon information is enriched through whole exon capture, transcriptome sequencing data with less interference between rRNA and genomic DNA, high expression enrichment of functional genes and low background is obtained, accordingly, requirements on RNA quality and initial amount are reduced, the step of removing rRNA interference is eliminated, and a more accurate and efficient way is provided for expressionchange analysis of mutant gene transcripts.

Description

Technical field [0001] The present invention relates to the field of biology, in particular to transcriptome data acquisition and analysis technology, more specifically, to a transcriptome sequencing method based on reverse transcription-whole exon capture technology (RT-WES). Background technique [0002] The transcriptome is the sum of all RNA that can be transcribed by a living cell during a certain developmental period or under certain physiological conditions. Transcriptome analysis is an important method to study cell phenotype and function. The current conventional transcriptome analysis methods mainly include hybridization-based biochip (including cDNA chip and oligonucleotide chip) technology and RNA sequencing technology based on sequence analysis. among them: [0003] Biochip technology is a semi-quantitative hybridization analysis technology, which can only analyze the level of expression of different gene transcripts, and cannot analyze the status of genome expressio...

AI Technical Summary

Problems solved by technology

Disadvantages: high requirements on sample RNA quality and initial quantity (requires RNA integrity RIN>7, initial quantity>2μg), need to use fresh tissue-derived RNA

Due to the difficulty of fresh tissue preservation, there are often no fresh tissue samples in clinical practice, only paraffin sections, and the RIN value of RNA obtained from paraffin sections is better than 3, and the poor ones cannot be evaluated at all. Therefore, for clinical samples, especially paraffin For RNA derived from sliced ​​white slices, it is impossible to directly build a library and obtain effective data using traditional RNA-seq technology. Even if more data is used for analysis, the effect is still not good, and the cost will increase a lot

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