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

A transcriptome sequencing and transcriptome technology, applied in the biological field, can solve the problems of inability to directly build a library and obtain valid data, high quality and input requirements, and difficulty in preserving fresh tissue, etc., to reduce further losses, low requirements, The effect of reducing quality and starting volume requirements

Inactive Publication Date: 2018-11-16
GENOMICARE BIOTECH SHANGHAI CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 bet

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Extract the total RNA of the sample

[0033] Use MagMAX produced by Thermofisher TM FFPE DNA / RNA Ultra Kit (Cat. No. A31881) kit, according to the instructions of the kit, extract RNA from formalin-fixed and paraffin-embedded (Formalin-Fixed and Parrffin-Embedded, FFPE) tissue samples to obtain FFPE samples Total RNA.

[0034] 2. Synthesis of cDNA

[0035] 1. First strand synthesis

[0036] 1) Take a 0.2ml PCR reaction tube without nuclease contamination, add the ingredients listed in Table 1 (wherein, the random primer contains 6 bases), pipette and mix. This step needs to be operated in a safety cabinet that removes nuclease contamination, and ensure that the water used is free of nuclease contamination.

[0037] Table 1

[0038] ingredient

Volume (μl)

Total RNA of FFPE sample (100-500ng)

13.5

Random primer (50ng / μl)

1

[0039] 2) Place the above PCR reaction tube on a PCR machine, incubate at 65°C for 5 minutes, and quickly transfer it to an ice box after comple...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2521/107C12Q2535/122
Inventor 王冠王晨魏金旺张敖戴春许强
Owner GENOMICARE BIOTECH SHANGHAI CO LTD
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