( Evelyn McGown Ph.D. and Michael S...)Dual-Luciferase Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #39)

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00

INTRODUCTION
Reporter gene assays are used to study eukaryotic gene expression. Dual geneticreporters are commonly used in transient transfections of cultured cells tominimize experimental variability caused by differences in cell number, viabilityor transfection efficiency. One vector containing the experimental reporter gene iscotransfected with a second vector containing a different reporter gene to serve asthe control. Typically, the experimental reporter is coupled to a regulatedpromoter, and the control reporter gene is coupled to a constitutive promoter. Bynormalizing the changes in expression of reporter activity to the activity of thecontrol, experimental variability can be minimized.

Colorimetric reporter assays have been widely used, but bioluminescent reportersystems, especially using firefly luciferase, are becoming increasingly popularbecause of their speed, sensitivity and wide dynamic range. PromegaCorporation has recently introduced their Dual-Luciferase Reporter Assay System which utilizes the firefly (Photinus pyralis) and Renilla (Renilla reniformis)luciferases¹. Because the enzymes have different substrate requirements, they canboth be measured in a single reaction mixture. The firefly luciferase is typicallyused as the experimental reporter, and the renilla luciferase serves as the control.

Figure 1 illustrates the two enzymatic reactions. Firefly luciferase enzymecatalyzes the oxidation of luciferin with the concomitant release of light². The reaction requires ATP, Mg⁺² and O₂. Renilla luciferase catalyzes the O₂-dependentoxidation of coelenterate luciferin (coelenterazine) but does not require Mg orATP³. Both reactions can easily be measured in the Molecular Devices Lmaxmicroplate luminometer.
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