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Method of constructing degradome sequencing library

A sequencing library and sequence technology, which is applied in the field of building a degradome sequencing library, can solve the problems of missing target genes, affecting the efficiency of target gene exploration, and the inability to distinguish the authenticity of predicted target genes, etc., and achieve the effect of simple operation

Inactive Publication Date: 2013-10-02
HANGZHOU LC BIOTECH
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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 of constructing degradome sequencing library
  • Method of constructing degradome sequencing library
  • Method of constructing degradome sequencing library

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Embodiment Construction

[0026] The technical scheme of the present invention is further described by taking the construction of the soybean degradome library as an example:

[0027] 1 Materials and reagents

[0028] The 3' adapter, 5' adapter and amplification primers were synthesized by Shanghai Sangon Company; the mRNA extraction kit, ligase and PCR kit were purchased from Thermo Fisher.

[0029] 2 methods and steps

[0030] 2.1 Extraction of mRNA from fresh soybean leaves

[0031] 2.1.1 Prepare 65°C and 80°C water baths;

[0032] 2.1.2 Dilute 100ug total RNA to 100ul with NF-H2O;

[0033] 2.1.3 After 5 minutes in a water bath at 65°C, place on ice;

[0034] 2.1.4 Take two 1.5ml spiral tubes and fill them with 50ul sera-mag oligo(dT)beads respectively;

[0035] 2.1.5 Wash the beads twice with 100ul Bead Binding buffer;

[0036] 2.1.6 Resuspend the beads with 100ul Bead Binding buffer, add 100ul total RNA;

[0037] 2.1.7 Gently flick for 5 minutes, place on the magnetic stand for 30 seconds, ...

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Abstract

The invention discloses a method of constructing degradome sequencing library. The method comprises following steps: sorting mRNA sections of miRNA target gene, connecting connectors of 5' terminals of mRNA sections of miRNA target gene, and amplifying cDNA of miRAN target gene. The technical scheme facilitates the operation, enables the specific loci where the animal and plant miRNAs act on the target gene to be located accurately, breaks through the bioinformatics prediction limitation, finds the target gene where miRNA acts on, and has the characteristics of efficiency, rapidness, intuition and accuracy.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to a method for constructing a degradome sequencing library Background technique [0002] miRNA (microRNA) is a type of endogenous non-coding RNA with a length of about 22 nucleotides, which is involved in almost all important life activities in animals and plants and regulates the expression of related genes. Usually, miRNA achieves the regulation of 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 the majority, and in plants The majority of target genes are cut. Differentially expressed miRNAs in animals and plants can be efficiently and quickly screened out using mature miRNA microarray chips or qPCR technology, and finding out the target genes of differential miRNAs becomes the key to studying their important biological functions. [0003...

Claims

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Application Information

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IPC IPC(8): C40B50/06
Inventor 郎秋蕾高晓莲周小川高威林彬金纯枝梁洪
Owner HANGZHOU LC BIOTECH
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