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Method for downregulating micro RNA-126 expression

An RNA-126, mir-126 technology, applied in the field of down-regulating the expression of microRNA-126, can solve the problems of complex construction, poor stability, and complicated technical operation

Inactive Publication Date: 2014-09-10
ZUNYI MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have the disadvantages of short action time, poor stability, complex construction, high requirements for instrument conditions, and complex technical operations, making it difficult to achieve the effect of conveniently, simply and effectively down-regulating the expression of miR-126

Method used

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Embodiment Construction

[0020] Embodiments of the present invention: the method for down-regulating the expression of microRNA-126,

[0021] 1) First retrieve the sequence of hsa-miR-126 in the miRBase database. The sense sequence of miR-126: 5'-CATTATTACTTTTGGTACGCGCTGTGACACTTCAAACTCGTACCGTGAGTAATAATGCG-3'; -126反义序列中CTCA转换成GGT;GTTT突变成CGCG,截取5'-CGCATTATTAGGTCGGTACGACGCG-3',把6个这样的相同片段串联起来,最终miR-126-sponge序列为5'-CGCATTATTAGGTCGGTACGACGCGCGCA TTATTAGGTCGGTACGACGCGCGCATTATTAGGTCGGTACGACGCGCGCATTATTAGGTCGGTACGACGCGCGCATTATTAGGTCGGTACGACGCGCGCATTATTAGGTCGGTACGA- 3', and finally add a HindⅢ restriction site at the 5' end, and a KpnI restriction site at the 3' end, and the miR-126-sponge sequence design is completed, such as figure 1 shown;

[0022] 2) Digest the backbone vector pEGFP-C2 and the target fragment miR-126-sponge with HindⅢ / KpnI double enzymes, recover the fragments and purify them, connect overnight at 16°C, transform after connection, pick 6 colonies, and inoculate them into cells containing 1...

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Abstract

The invention discloses a method for downregulating micro RNA-126 expression, which is characterized in that a molecular biology technology is used, on a base of an eukaryotic vector containing a CMV promoter, an eukaryotic expression carrier containing miR-126-sponge is constructed, then RNA-126 expression is downregulated; the carrier enables in-vitro transfection to the eukaryotic cells, a fluorescence quantification PCR instrument is used for detecting the expression of a miR-126 mature form, and the biology effect after downregulating miR-126 expression can be observed through in-vitro experiment simultaneously. The method is capable of conveniently and rapidly downregulating miR-126 expression with long-acting, and can rapidly observe the biology effect after downregulating miR-126 expression. The method is adapted to cell transfection, and can be used for preparing the transgene animals.

Description

technical field [0001] The invention relates to the field of life sciences, in particular to a method for down-regulating the expression of microRNA-126. Background technique [0002] MicroRNA-126 (microRNA-126, miR-126) has important biological roles. At present, the techniques for changing the expression of miR-126 to study its biological function mainly include miR-126 sponge (Sponge) expressed by lentiviral vector, antisense oligonucleotides (ASO), inhibitor (Inhibitor) and Antagomir wait. However, these methods have the disadvantages of short action time, poor stability, complex construction, high requirements for instrument conditions, and complex technical operations, making it difficult to achieve the effect of conveniently, simply and effectively down-regulating the expression of miR-126. Contents of the invention [0003] The purpose of the present invention is to provide a method for down-regulating the expression of microRNA-126, which is simple in design and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12N15/79
Inventor 徐林胡燕周涯李永菊廖珍媛
Owner ZUNYI MEDICAL UNIVERSITY
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