Method for screening miR-181b target gene

A mir-181b, target gene technology, applied in the field of molecular biology

Inactive Publication Date: 2018-09-14
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) miRNA perfectly matches and complements the target gene mRNA, degrades the target gene, thereby inhibiting gene expression

Method used

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  • Method for screening miR-181b target gene
  • Method for screening miR-181b target gene
  • Method for screening miR-181b target gene

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Embodiment Construction

[0065] This embodiment provides a method for screening chi-miR-181b target gene, comprising the following steps:

[0066] 1) Using goat ovary tissue cDNA as template, in Taq DNA polymerase, buffer environment, Mg 2+ 1. In the presence of dNTPs, use the primer RUNX1 to amplify under PCR conditions, and determine that the obtained PCR product is the sequence of the 3'UTR region of the RUNX1 gene;

[0067] The primer RUNX1 is as follows (the underline is the restriction site of XhoI and NotI endonuclease):

[0068] Upstream primer F: 5'-CG CTCGAG AGATCAGTATTGACGCTGATCA-3';

[0069] Downstream primer R: 5'-AT GCGGCCGC CATCCAGAATATCACAAATAACC-3'.

[0070] The condition of described PCR amplification is:

[0071] 15 μL reaction system, including 0.5 μL DNA template, 7.5 μL MasterMix, 0.5 μL upstream and downstream primers, add sterilized water to 15 μL;

[0072] Described PCR amplification reaction procedure is as follows:

[0073] The PCR reaction program using primer RUNX1...

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Abstract

The invention discloses a method for screening a miR-181b target gene. The method comprises the following steps: taking a goat ovarian tissue cDNA (complementary Deoxyribonucleic Acid) as a template and carrying out amplification in the presence of Taq DNA polymerase, a buffering environment, Mg<2+> and dNTPs by utilizing a primer RUNX1 under the condition of PCR (Polymerase Chain Reaction), so asto obtain a determined PCR product which is a sequence of a 3' UTR region of an RUNX1 gene; then constructing a dual-luciferase reporting system and detecting the activity of luciferase; primarily identifying the miR-181b target gene; detecting the influences, caused by the fact that miR-181b is detected by adopting an RT-qPCR method, on the level of an RUNX1 gene on mRNA (massager Ribonucleic Acid); detecting the influences, caused by the fact that the miR-181b is detected by adopting a Western blot method, on a protein level of the RUNX1 gene. A combination site with the miR-181b exists inthe RUNX1 3' UTR region; a modern molecular biotechnology is used for verifying a targeting regulation relation of the miR-181b and the target gene RUNX1 and the miR-181b can be used for inhibiting the expression of the RUNX1 gene in the mRNA and protein levels; furthermore, the method confirms that the RUNX1 is a target gent of the miR-181b. The method lays a foundation for further researching influences, caused by the miR-181b, on ovarian development and lambing performance of dairy goats.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to molecular cloning, RT-qPCR and Western blot detection technologies, and in particular to a method for screening miR-181b target genes, mainly by constructing a dual-luciferase reporter carrier and using modern molecular biotechnology to explore And verify the target gene of miR-181b. Background technique [0002] miRNA is a short (21-23 nucleotides), evolutionarily conserved, non-coding RNA molecule that has the function of post-transcriptional regulation of gene expression. It was first discovered in Caenorhabditis elegans, and it can Binding of the 3' non-coding (UTR) of the target gene inhibits translation of mRNA into protein. The regulation of miRNA is quite complex, each miRNA can regulate multiple target genes through different signaling pathways, and each target gene may be regulated by several miRNAs simultaneously. As an important regulatory factor, miRNA widely exists ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66C12Q1/6851G01N33/68
CPCC12Q1/66C12Q1/6851G01N33/68
Inventor 曹斌云安小鹏宋宇轩李广高可心
Owner NORTHWEST A & F UNIV
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