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Novel method for reducing miRNA-21 expression

A technology of mirna-21 and mir-21-aso, applied in DNA/RNA fragments, recombinant DNA technology, using vectors to introduce foreign genetic material, etc., can solve the problems of short effective time and difficult operation

Inactive Publication Date: 2015-03-11
ZUNYI MEDICAL UNIVERSITY
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Problems solved by technology

[0003] The purpose of the present invention is to provide a new method for down-regulating the expression of miRNA-21, which solves the problems of short effective time and difficult operation of the existing methods for changing the expression of miRNA-21

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  • Novel method for reducing miRNA-21 expression

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Embodiment Construction

[0014] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0015] The invention utilizes molecular cloning and antisense nucleic acid technology to construct a pcDNA-6.2-miR-21-ASO eukaryotic expression vector containing a CMV promoter. After the vector was transfected into eukaryotic cells in vitro, the expression of miR-21 was detected by fluorescent quantitative PCR technology, and the biological effect of down-regulated miR-21 was observed through in vitro experiments. The invention belongs to the field of biotechnology and can be widely used in molecular biology and cell biology experiments to down-regulate miR-21 and observe its biological effects.

[0016] The present invention adopts following steps to carry out:

[0017] Step 1: miR-21-ASO sequence design: retrieve the >miR-21-5p sequence in the miRBase database: 5'-UAGCUUAUCAGACUGAUGUUGA-3'; its antisense complementary sequence is: 5'-TCAA...

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Abstract

The invention discloses a novel method for reducing miRNA-21 expression. The method comprises the following steps: retrieving the sequence of miR-21-5p from a miRBase database, that is, 5'-UAGCUUAUCAGACUGAUGUUGA-3', wherein an antisense complementary sequence of the >miR-21-5p is 5'-TCAACATCAGTCTGATAAGCTA-3', then, complementarily connecting with a sticky end of a carrier so as to obtain synthetized positive-sense and antisense strands; mixing the synthetized positive-sense and antisense strands, heating the mixed strands at the temperature of 95 DEG C for 5 minutes and then annealing so as to form double strands; connecting the annealed double chains DNA at the room temperature for 30 minutes so as to obtain a connected product; taking 2-10 [mu]l of the connected product, transforming the product into 100 [mu]l of competence cells DH5 alpha, coating an LB flat plate, wherein 50 [mu]g / ml of spectinomycin is contained, performing incubation at the temperature of 37 DEG C for 16 hours, taking 6 positive clones, innoculating the clones to an LB culture medium containing 50 [mu]g / ml of spectinomycin, and extracting plasmids by using a Tiangen kit for a small amount of plasmid extraction. The novel method disclosed by the invention has the benefit that the miR-21 expression can be reduced in a long-term and convenient manner.

Description

technical field [0001] The invention belongs to the technical field of biological genes and relates to a novel method for down-regulating the expression of miRNA-21. Background technique [0002] MicroRNA-21 (microRNA-21, miR-21) is one of the members of the miRNA family and has important biological functions. At present, the methods for studying the biological functions of miR-21 by changing the activity or expression mainly include inhibitors (Inhibitor), antagonists (Antagomir) and lentiviral vectors. However, these methods have shortcomings such as short effective time, difficult operation, and poor safety performance, making it difficult to achieve long-acting and convenient down-regulation of miR-21 expression. Contents of the invention [0003] The purpose of the present invention is to provide a novel method for down-regulating the expression of miRNA-21, which solves the problems of short effective time and difficult operation of the existing method for changing ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113
Inventor 徐林陶弋婧周涯郭萌萌赵娟娟
Owner ZUNYI MEDICAL UNIVERSITY
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