Kit for identifying miRNA (micro-ribonucleic acid) target genes and applications thereof

A reporter gene, coding gene technology, applied in the field of genetic engineering, can solve problems such as cumbersome detection

Active Publication Date: 2013-06-05
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are relatively cumbersome

Method used

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  • Kit for identifying miRNA (micro-ribonucleic acid) target genes and applications thereof
  • Kit for identifying miRNA (micro-ribonucleic acid) target genes and applications thereof
  • Kit for identifying miRNA (micro-ribonucleic acid) target genes and applications thereof

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Experimental program
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Embodiment 1

[0037] Embodiment 1, the construction of reporter gene carrier system

[0038] The cloning vector pMD18-T is the product of TAKARA Company, the vector pGL3-Basic, the internal reference vector pRL-TK vector and the dual reporter gene detection reagent are all products of Promega Company; Escherichia coli competent DH5α is produced by Beijing Biomed Biotechnology Company; The HepG2 line (purchased from the Basic Medical Cell Center of Peking Union Medical College) was preserved and provided by our laboratory; the miRNA extraction kit was miRNAeasy mini kit from QIAGEN Company, and the Reverse Transcriptase M-MLV (RNase H - ), primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0039] 1. Construction and identification of experimental reporter vector pMIR-Luciferase

[0040] 1. Construction of pCMV-luciferase vector

[0041] ① Design CMV promoter primers as shown in Table 1, use pcDNA3.1+ (purchased from Invitrogen Company No. V790-20.) as a template, and use CMV p...

Embodiment 2

[0056] Embodiment 2, functional verification of the reporting system

[0057] 1. Construction of pMIR-CFHUTR vector:

[0058] ①Synthesize primers according to Table 1; extract RNA from HepG2 cells, reverse transcribe mRNA with oligo dT primers as templates, use CFH-3′UTR amplification upstream primers and CFH-3′UTR amplification downstream primers as primers, and perform PCR Amplified, and the obtained PCR product was sent for sequencing, and the result was that the PCR product had the nucleotide shown in sequence 4 in the sequence listing (the nucleotide in the 3'-UTR region of CFH). Electrophoretic detection such as figure 2 As shown in the left figure, a 247bp fragment was obtained, and the PCR product was connected to the pMD18T vector. After transformation, a transformant was obtained. The plasmid of the transformant was extracted and sent for sequencing. The result was that the plasmid was obtained by inserting sequence 4 in the sequence table into the pMD18T vector. ...

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Abstract

The invention discloses a kit for identifying miRNA (micro-ribonucleic acid) target genes and applications thereof, namely the application of a report vector in identification of miRNA target genes and / or detection of miRNA expression, the application of the kit in the identification of the miRNA target genes and / or the detection of the miRNA expression, and the application of the vector in the identification of the miRNA target genes and / or the detection of the miRNA expression, wherein the report vector comprises report genes, as well as a promoter and multiple cloning sites, which are positioned at the up stream and down stream of the report genes respectively, and the report genes are genes encoded by firefly luciferase. Experiments prove that the research designs a dual-report gene vector system for identification of the miRNA target genes, which is used for regulating and controlling fast identification of the target genes and evaluating the regulation and the control of the miRNA expression in the miRNA research.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a kit for identifying miRNA target genes and applications thereof. Background technique [0002] A class of non-coding protein RNA genes widely exist in animal and plant genomes, producing RNAs with a length of about 18-25 nucleotides, which are named miRNA (microRNA), and they can regulate genes through post-transcriptional or translational levels Express. Participate in the regulation of cell development, neural differentiation, cell proliferation, apoptosis and fat metabolism. [0003] The primary transcription product of miRNA, pri-miRNA, can be processed by RNase III into the precursor pre-miRNA of miRNA, and the pre-miRNA formed in the nucleus can pass through the nuclear membrane from the nucleus to the cytosol, and be cleaved by RNase III nucleases to form A 21nt single-stranded miRNA that forms a mature miRNA. Mature miRNAs enter the RISC complex and regulate gene e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/66C12N15/63
Inventor 李军锋周育森鲍春旸于虹郭彦
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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