Factors modulating expression of Renilla luciferase from control plasmids used in luciferase reporter gene assays

Abstract

The Renilla luciferase gene is commonly used as an internal control in luciferase-based reporter gene assays to normalize the values of the experimental reporter gene for variations that could be caused by transfection efficiency and sample handling. Various plasmids encoding Renilla luciferase under different promoter constructs are commercially available. The validity of the use of Renilla luciferase as an internal control is based on the assumption that it is constitutively expressed in transfected cells and that its constitutive expression is not modulated by experimental factors that could result in either the upregulation or the downregulation of the amounts of the enzyme produced. During the past ten years, a number of reports have appeared that identified a variety of conditions that could alter the basal constitutive expression of Renilla luciferase. The use of Renilla luciferase in those circumstances would not be valid and an alternative way of normalization would be necessary. This review covers the factors that have been reported thus far as modulating the expression of Renilla luciferase from plasmid constructs.

Zinc ions upregulate the hormone gastrin via an E-box motif in the proximal gastrin promoter

“…Briefly, cells were lysed with 60-70 ml/well Reporter Lysis Buffer, and Firefly and Renilla luciferase activity were measured with a FLUOstar Optima plate reader (BMG Labtech, Victoria, Australia). As Renilla luciferase activity from the control vector pRL-TK is induced by zinc (data not shown), the use of Renilla luciferase would not be valid in assays where cells were treated with zinc and in these experiments relative luciferase activity was therefore normalized to the total protein content of each well (Shifera & Hardin 2010) as determined with a Bradford protein assay kit (Bio-Rad). Whenever possible Firefly luciferase activity was normalized to Renilla luciferase activity.…”

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

“…The specific readout of a reporter assay is normalized as firefly luciferase activity (test gene) over Renilla luciferase activity (internal control gene). It is imperative to establish that internal control reporters do not respond to the experimental conditions because this can inappropriately distort the readout ratio (429).…”

AP4 Transcription Factor Binding Site is a Repressor Element in ek2 Promoter of Human Liver Carcinoma Cell Line, HepG2

“…Firefly luciferase encoded by luc2 gene in pGL4.10 vector generates luminescence in the green-yellow region (550-570 nm) from beetle luciferin in the presence of ATP, magnesium and oxygen (de Wet et al 1987;Thorne et al 2010). Renilla (Renilla reniformis) on the other hand catalyzes the oxidation of coelenterazine to produce coelenteramide, carbon dioxide and emits blue light of 480 nm (Shifera & Hardin 2010). Renilla luciferase uses a different substrate from firefly luciferase and its independence of ATP and magnesium usage making it suitable as the internal control in the dual reporter system.…”