采用体外细胞培养荧光素酶试验法测试MicroRNA靶标

Nathalie Coré

doi:10.21769/BioProtoc.420

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

发布: 2013年03月05日第3卷第5期 DOI: 10.21769/BioProtoc.420 浏览次数: 16467

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Nature Neuroscience, featuring study using the protocol.

Aug 2012

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

哺乳动物细胞中利用基于分析流式细胞术的双荧光报告因子研究miRNA诱导调控

Nicolas Lemus-Diaz [...] Jens Gruber

2018年09月05日 13109 阅读

小鼠体内siRNA转染CD40沉默和肾免疫染色评价

Miguel Hueso [...] Joan Torras

2021年05月20日 10570 阅读

原代培养皮层神经元中高效 siRNA 转染方法

Xiaorong Wang [...] Zhaolong Zhang

2026年01月20日 905 阅读

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)

3'UTR (3’UTR)

Luciferase assay (荧光素酶检测)

In vitro (体外)

Materials and Reagents

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