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mRNA nucleoplasmic ratio variation-based method for identifying miRNA target gene and application thereof

An identification method and target gene technology, which can be used in biochemical equipment and methods, microbial determination/inspection, material inspection products, etc., and can solve problems such as miRNA discovery

Inactive Publication Date: 2011-02-02
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unfortunately so far there is no experimental method that can quickly, efficiently and accurately discover the targets of miRNAs.

Method used

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  • mRNA nucleoplasmic ratio variation-based method for identifying miRNA target gene and application thereof
  • mRNA nucleoplasmic ratio variation-based method for identifying miRNA target gene and application thereof

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Embodiment Construction

[0012] The following figure further describes the present invention by identifying the functional target gene of miR-200b in HeLa cells through specific application of the technical method of the present invention.

[0013] 1. Bioinformatics prediction:

[0014] Bioinformatics software such as TargetScan, miRanda, DIANAmicroT, PicTar, RNAHybrid, miRGen++, MiTarget, TarBase and other bioinformatics software were comprehensively used to predict the potential target genes of miR-200b, and the genes with the highest scores were randomly selected as targets ( figure 1 ).

[0015] 2. HeLa cytoplasmic and nuclear RNA extraction:

[0016] The cytoplasmic / nuclear RNA extraction kit (Norgen) was used to extract RNA from HeLa cells, and the main steps were as follows:

[0017] 1) Collect 3x10 6 Wash the cells once with ice-cold PBS, add 200 μl ice-cold lysate, and mix for 15 seconds.

[0018] 2) After the lysis is complete, centrifuge at room temperature at 16,000 rpm for 3 minutes, ...

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Abstract

The invention belongs to the technical field of biology, and relates to a rapid and accuracy message Ribonucleic Acid (mRNA) nucleoplasmic ratio variation-based method for identifying a mirna Ribonucleic Acid (miRNA) target gene, and to application of the method in the biomedical field. The method is realized through the following technical scheme of: predicting possible target genes of a certain miRNA through the conventional miRNA target prediction bioinformatics software, such as Targetscan; measuring the mRNA nucleoplasmic ratio variation of the possible target genes in a cell by utilizing a biochip or real-time quantitative Polymerase Chain Reaction (PCR); searching the possible target genes with an mRNA nucleoplasmic ratio of more than 1; and further confirming in the protein level through Western blot.

Description

Technical field: [0001] The invention relates to a rapid and accurate identification method of miRNA target gene based on the change of mRNA mass-to-nucleus ratio; it relates to the application of the method in the field of biomedicine. Background technique: [0002] MicroRNA, referred to as miRNA, is a 21-25 base non-coding small molecule RNA, which widely exists in eukaryotes. The primary transcription product (pri-miRNA) of the miRNA gene is cleaved by RNaseIII Drosha in the nucleus to become a precursor miRNA (pre-miRNA), and the pre-miRNA is transferred from the nucleus to the cytoplasm under the action of the transport protein 5 (exportin5). The mature miRNA is then further cleaved by another RNaseIII Dicer. These mature miRNAs form a RISC (RNA-induced silencing complex) complex with other proteins, thereby causing degradation or translational inhibition of target mRNAs. A large number of studies have shown that miRNA is involved in many important biological processe...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12Q1/68
Inventor 邵宁生李洁黄宝春夏伟陈留存李少华苏雪婷王芳丁红梅
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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