Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone.
The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.
Ibrahim
N M
NM
Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA. nibrahim@physio.emory.edu
Marinovic
A C
AC
Price
S R
SR
Young
L G
LG
Fröhlich
O
O
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003907:Dexamethasone / Q000494:pharmacology*; D013196:Dihydrotestosterone / Q000494:pharmacology*; D008156:Luciferases / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D013196:Dihydrotestosterone; D003907:Dexamethasone; D008156:Luciferases; D013937:Thymidine Kinase