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A method for screening mir-3880 target genes

A technology of mir-3880 and screening methods, applied in the direction of biochemical equipment and methods, other methods and instruments for inserting foreign genetic materials

Active Publication Date: 2021-05-11
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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  • A method for screening mir-3880 target genes
  • A method for screening mir-3880 target genes
  • A method for screening mir-3880 target genes

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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 miR-3880 target gene screening, which uses goat genome DNA as a template, in the presence of Taq DNA polymerase, buffer environment, Mg 2+ In the presence of dNTPs, the primer OGR1 was used to amplify under PCR conditions, and the obtained PCR product was determined to be the sequence of the 3'UTR region of the OGR1 gene; then a dual-luciferase reporter system was constructed to detect luciferase activity and preliminary identification The target gene of miR-3880; RT-qPCR was used to detect the effect of miR-3880 mimics on the OGR1 gene at the mRNA level; Western blot was used to detect the effect of miR-3880 mimics on the OGR1 gene at the protein level. It was clarified that there is a binding site for miR-3880 in the 3'UTR region of OGR1, and the targeting regulatory relationship between miR-3880 and the target gene OGR1 was verified. miR-3880 can inhibit the expression of OGR1 gene at the mRNA and protein levels, further confirming OGR1 is a target gene of miR‑3880. It laid a foundation for studying the effect of miR‑3880 on the mammary gland development and lactation function 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 Patents(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