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Method for screening miR-3880 target gene

A technology of mir-3880 and screening methods, applied in the direction of biochemical equipment and methods, other methods of inserting foreign genetic materials, material inspection products, etc.

Active Publication Date: 2018-09-07
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-3880 target gene
  • Method for screening miR-3880 target gene
  • Method for screening miR-3880 target gene

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Embodiment Construction

[0067] This embodiment provides a miR-3880 target gene screening method, comprising the following steps:

[0068] 1) Using goat genomic DNA as a template, in Taq DNA polymerase, buffer environment, Mg 2+ 1. In the presence of dNTPs, use primer OGR1 to amplify under PCR conditions, and determine that the obtained PCR product is the sequence of the 3'UTR region of the OGR1 gene;

[0069] The primer OGR1 is as follows (the underline is the restriction site of XhoI and NotI endonuclease):

[0070] Upstream primer F: 5'-CG CTCGA GGTGGAGGTTGGATGGGGAGA-3';

[0071] Downstream primer R: 5'-AT GCGGCCGC ACACATGCACACACAGAGCC-3'.

[0072] The condition of described PCR amplification is:

[0073] The 15 μl reaction system includes: 0.5 μl DNA template, 7.5 μl MasterMix, 0.5 μl each of upstream primer F and downstream primer R of primer OGR1, add sterilized water to 15 μl;

[0074] Described PCR amplification reaction procedure is as follows:

[0075] The PCR reaction program using ...

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Abstract

The invention discloses a method for screening a miR-3880 target gene. The method comprises the following steps of using a goat genome DNA (deoxyribonucleic acid) as a template, utilizing a primer OGRI to perform amplification by PCR (polymerase chain reaction) under the existence of Taq DNA polymerase, buffer environment, Mg<2+> and dTNPs, and determining an obtained PCR product is a sequence ofa 3'UTR zone of the gene OGR1 (ovarian cancer G protein-coupled receptor 1); constructing a double-luciferase report system, detecting the activity of luciferase, and primarily identifying the miR-3880 target gene; adopting a RT-qPCR (real-time and quantitative polymerase chain reaction) method to detect the influence on the mRNA level of the gene OGR1 by an miR-3880 simulation matter; adopting aWestern blot method to detect the influence on the protein level of the gene OGR1 by the miR-3880 simulation matter. The method has the advantages that the existence of the 3'UTR zone of the gene OGR1and the binding sites of the miR-3880 are defined, the targeting regulating and control relationship between the miR-3880 and the target gene OGR1 is verified, the miR-3880 can be used for inhibitingthe expression of the gene OGR1 on mRNA and protein levels, and the gene OGR1 is further verified to be the miR-3880 target gene; a foundation is laid for the study on the influence on the mammogenesis and lactation functions of dairy goats.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to molecular cloning, RT-qPCR and Western blot detection technology, and specifically relates to a miR-3880 target gene screening method, mainly by constructing a dual-luciferase reporter carrier, using modern molecular biological technology to explore and Validation of target genes of miR-3880. 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 ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/88C12Q1/6897C12Q1/6851G01N33/68
CPCC12N15/85C12N15/88C12Q1/6851C12Q1/6897G01N33/68G01N2333/726C12Q2531/113C12Q2545/101
Inventor 安小鹏曹斌云宋宇轩李广杜晓岩
Owner NORTHWEST A & F UNIV
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