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Method for constructing degradome sequencing library

A sequencing library and sequencing technology, applied in the field of genetic engineering, can solve the problems of missing target genes, inability to distinguish the authenticity of predicted target genes, affecting the efficiency of target gene exploration, etc. Effect

Pending Publication Date: 2018-09-25
杭州联川基因诊断技术有限公司
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Problems solved by technology

However, since the prediction method cannot distinguish the authenticity of the predicted target gene, all prediction results must be experimentally confirmed to eliminate the false and preserve the true.
This greatly affects the discovery efficiency of target genes
At the same time, in some cases, such as when there is a high mismatch between the miRNA and the target gene, the prediction method may miss many real target genes

Method used

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  • Method for constructing degradome sequencing library
  • Method for constructing degradome sequencing library
  • Method for constructing degradome sequencing library

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Embodiment Construction

[0020] The technical scheme of the present invention will be further specifically described below by taking the construction of a soybean leaf degradome library as an example.

[0021] 1. Materials and reagents

[0022] mRNA Capture Beads extraction kit was purchased from Vazyme Company; RNase Inhibitor was purchased from Promega Company; 5'Adapter B, reverse transcription kit and PCR kit were purchased from Wujiang Huijie Company.

[0023] 2. Operation process

[0024] A. Extraction of soybean fresh leaves mRNA

[0025] 1. Prepare 65°C and 80°C water baths;

[0026] 2. Take out the mRNA Capture Beads from 4°C and let it stand to allow the temperature to equilibrate to room temperature;

[0027] 3. Add 20μg total RNA to 50μL with Nuclease-free water;

[0028] 4. Add the well-mixed mRNA Capture Beads to the total RNA sample;

[0029] After 5 minutes in a water bath at 5.65°C, place at 4°C to denature the RNA;

[0030] 6. Gently flick for 5 minutes at room temperature, pla...

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Abstract

The invention discloses a method for constructing a degradome sequencing library. The method comprises the following steps: the sorting of mRNA fragments of a miRNA target gene; the ligation of 5' terminal adapters with the mRNA fragments of the miRNA target gene; reverse transcription with random primers; and the amplification of the cDNA of the miRNA target gene. According to a technical schemeof the invention, ligation of specific 3' terminal adapters is not needed, so operation is simple, little time is needed, and requirements on a sample size are greatly reduced; the method can accurately find out specific sites where animal and plant miRNAs act on target genes, get rid of the limitation of bioinformatics prediction, and actually find out target genes on which miRNA acts through experiments; and the method is characterized in that the method is efficient, fast, visual and accurate.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a method for constructing a degradome sequencing library. Background technique [0002] miRNA (microRNA) is a kind of endogenous non-coding RNA with a length of about 22 nucleotides, which can regulate the expression of related genes and participate in almost all important life activities in animals and plants. Usually, miRNA regulates gene expression by partially or completely pairing with the target gene (mRNA), causing gene translation inhibition or gene endonuclease cleavage. In animals, gene translation inhibition is mostly, while in plants, it is Most of the target genes are spliced. Differentially expressed miRNAs in animals and plants can be efficiently and quickly screened by mature miRNA microarray chip or qPCR technology, and the identification of target genes regulated by miRNAs is the key to elucidating their complex regulatory mechanisms...

Claims

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Application Information

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IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 郎秋蕾周小川高威林彬金纯枝梁洪
Owner 杭州联川基因诊断技术有限公司
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