A convenient plasmid-based system containing three reporter genes for real-time and quantitative analysis of messenger RNA silencing.
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.
Feng
Liqiang
L
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.
Li
Feng
F
Liu
Yichu
Y
Zheng
Xuehua
X
Zhang
Biliang
B
Chen
Ling
L
D002460:Cell Line; D003199:Computer Systems; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D049409:Luciferases, Firefly / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection
D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly