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Test MicroRNA Target with in vitro Cell Culture Luciferase Assay

采用体外细胞培养荧光素酶试验法测试MicroRNA靶标

NC Nathalie Coré

发布: 2013年03月05日第3卷第5期 DOI: 10.21769/BioProtoc.420 浏览次数: 15674

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Abstract

MicroRNAs (miRNAs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. This protocol is to be used to test the binding and activity of miRNA on putative UTR target sequences. It is based on the expression of Luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miRNA of interest or synthetic miRNA to test in an in vitro cell culture assay.

Keywords: MicroRNA activity (microRNA活性) search 3'UTR (3’UTR) search Luciferase assay (荧光素酶检测) search In vitro (体外) search

Materials and Reagents

  1. HEK293T cells (ATCC, catalog number: CRL-11268 TM)
  2. DMEM (High Glucose) (GutaMAXTM) (Life technologies, catalog number: 61965 )
  3. Fetal bovine serum (FBS) heat inactivated (Sigma-Aldrich, catalog number: F9665 )
  4. Penicillin-Streptomycin (Life technologies, catalog number: 15140 )
  5. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, catalog number: E1330 )
  6. miScript miRNA Mimic (QIAGEN, catalog number varies depending on your interest miRNA)
  7. Lipofectamine®2000 Transfection Reagent (Life Technologies, catalog number: 11668 )
  8. Dual-Glo Luciferaseassay system (PromegaCorporation, catalog number: E2920 )
  9. Cuiture medium (see Recipes)

Equipment

  1. Luminometer (Berthold Technologies)
  2. White 96-well plates (Greiner, catalog number: 655074 )

Procedure

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文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Coré, N. (2013). Test MicroRNA Target with in vitro Cell Culture Luciferase Assay. Bio-protocol 3(5): e420. DOI: 10.21769/BioProtoc.420.

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分类

分子生物学 > RNA > RNA 干扰

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