一种便捷的基于质粒的系统，包含三个报告基因，用于信使核糖核酸沉默的实时定量分析。

A convenient plasmid-based system containing three reporter genes for real-time and quantitative analysis of messenger RNA silencing.

作者信息

Feng Liqiang, Li Feng, Liu Yichu, Zheng Xuehua, Zhang Biliang, Chen Ling

机构信息

Center for Vaccines and Biotherapeutics, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou International Business Incubator A3, Guangzhou Science City, Guangzhou 510663, China.

出版信息

Anal Biochem. 2009 Nov 15;394(2):284-6. doi: 10.1016/j.ab.2009.07.031. Epub 2009 Jul 25.

DOI:10.1016/j.ab.2009.07.031

PMID:19635448

Abstract

Luciferase genes have been used extensively for quantitative analysis in RNA interference (RNAi) and endogenous microRNA (miRNA) studies. However, one drawback is that determination of luciferase activity always requires that cells be killed, allowing less real-time information about a biological process to be obtained. Here we describe a triple-reporter plasmid for target miRNA analysis in which enhanced green fluorescent protein (EGFP) and Renilla luciferase (RLuci) are linked by "self-cleave" 2A under control of the CMV promoter. Firefly luciferase (FLuci) serves as internal control under control of another independent promoter. Our real-time system provides a convenient and improved approach for assessing messenger RNA silencing in vivo.

摘要

荧光素酶基因已广泛用于RNA干扰（RNAi）和内源性微小RNA（miRNA）研究中的定量分析。然而，一个缺点是荧光素酶活性的测定总是需要杀死细胞，从而减少了关于生物过程的实时信息。在这里，我们描述了一种用于靶miRNA分析的三报告质粒，其中增强型绿色荧光蛋白（EGFP）和海肾荧光素酶（RLuci）在巨细胞病毒（CMV）启动子的控制下通过“自切割”2A连接。萤火虫荧光素酶（FLuci）作为另一个独立启动子控制下的内参。我们的实时系统为评估体内信使RNA沉默提供了一种方便且改进的方法。