Method for rapidly identifying shRNA effectiveness

An effective and fast technology, applied in the field of molecular biology, can solve problems such as high cost, long time, and high risk of biological safety, and achieve the effect of reducing workload, maintaining consistency, and reducing labor costs

Pending Publication Date: 2021-02-02
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is: how to provide a method for quickly identifying the effectiveness of shRNA, and solve the problems of high biological safety risk, long time and high cost of existing identification methods

Method used

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  • Method for rapidly identifying shRNA effectiveness

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Experimental program
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Effect test

Embodiment

[0021] 1. Design and synthesize multiple shRNA sequences as candidate functional shRNAs according to the target gene sequence and the shRNA design rules of the PLKO.1 vector;

[0022] Specific implementation steps: log in to the shRNA design website, enter the name of the target gene, and search for candidate siRNA sequence fragments of the target gene. After the sequence specificity was verified by the blast comparison tool in the NCBI database, according to the design rules of PLKO.1shRNA (Forward oligo: 5'CCGG—21bpsense—CTCGAG—21bpantisense—TTTTTG3', Reverse oligo:5'AATTCAAAAA—21bp sense—CTCGAG—21bp antisense3') to complete the design of shRNA sequence fragments and send them to chemical synthesis.

[0023] 2. According to the vector construction steps, first dissolve the primers and configure them into an annealing reaction system, heat them in a constant temperature water bath at 95°C, and then cool and anneal naturally to form shRNA fragment dimers. At the same time, Ec...

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Abstract

The invention discloses a method for rapidly identifying shRNA effectiveness. The method comprises the following steps: (1) designing and synthesizing a plurality of candidate shRNAs as candidate functional shRNAs; (2) constructing a PLKO.1-shRNA recombinant vector based on a lentiviral vector; (3) in HEK293/HEK293T cells, co-transfecting the HEK293/HEK293T cells into the cells by using a cell transient transfection method; (4) after the transfection is finished for 6-8 hours, replacing a fresh culture medium for the cells and continuously culturing for 24-48 hours; (5) collecting the cells, cracking the cells, and extracting total protein; and (6) detecting the expression of a target protein by Western Blot, and identifying the effectiveness of the candidate shRNAs by comparing the expression difference of target proteins in a control group and a treatment group. According to the method, the time cost is saved by about 50%, the overall experimental workload is reduced by about 70%, and the labor cost is remarkably and greatly reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for quickly identifying the effectiveness of shRNA. Background technique [0002] The realization of target gene silencing based on RNA interference technology is one of the important application bases of modern life science and genetic engineering operations. There are two main approaches to achieve RNAi-based targeted gene silencing at the cellular level. 1. Target gene silencing mediated by siRNA transient transfection; 2. Target gene silencing mediated by shRNA lentiviral packaging infected cells; among them, the second method can achieve stable target gene silencing, and is suitable for different cells All types can be effectively applied and thus have a wider range of applications. [0003] The sequence design of shRNA must be based on the sequence sequence of the target gene itself, and multiple shRNA sequences can be designed for the same target pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12Q1/68G01N33/68
CPCC12N15/86C12N15/65C12Q1/68G01N33/68C12N2740/15043C12N2800/107C12Q2525/207
Inventor 张昊星李延鹏李嘉恒吴精博应明
Owner SHENZHEN UNIV
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