Promega Notes Magazine Number 57, 1996, p.02 Dual-Luciferase TM Reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays By Bruce A. Sherf, Shauna L. Navarro, Rita R. Hannah and Keith V. Wood Promega Corporation In the quantitation of gene expression using firefly luciferase, a second reporter gene is commonly used to minimize experimental variability. However, traditional co-reporters (e.g., CAT, beta-Gal or GUS) are inconvenient due to differences in their respective assay chemistries, handling requirements and measurement characteristics. Promega introduces a superior dual-reporter technology integrating the assay of firefly luciferase with the Renilla luciferase assay. The Dual-Luciferase TM Reporter Assay System*, in combination with the pRL Vectors which express Renilla luciferase as the second reporter, offers the exceptional speed, sensitivity and convenience of two luciferase reporter assays in a single-tube format. The system also includes Passive Lysis Buffer, formulated to provide quantitative solubilization of both luciferases from mammalian cells cultured in multi-well plates without the need for individual processing of each sample. The Dual-Luciferase TM Reporter Assay System will provide immediate benefit to researchers currently using any experimental firefly luciferase reporter vector. *Patent pending. Introduction Dual-reporters are used to make relational or ratiometric measurements within an experimental system. Typically one reporter is used as an internal control to which measurement of the other reporter is normalized. In measurements of gene expression, dual reporters are generally used in transient transfections of cultured cells, where one vector containing the experimental reporter gene is co-transfected with a second vector containing a distinct reporter gene serving as the control. Usually, the experimental reporter is coupled to a regulated promoter to study the structural or physiological basis of regulated gene expression. Relative changes in the expression of reporter activity correlate to changes in the transcriptional activity of the coupled regulated promoter. To provide an internal control for transcriptional activity, the second reporter gene is coupled to a constitutive promoter that is unperturbed by the various experimental conditions. By this method, it is possible to minimize inherent variabilities that can undermine experimental accuracy, such as differences in the number and health of the cultured cells, and the efficiencies of cell transfection and lysis. Dual-reporter applications utilizing firefly luciferase in combination with either chloramphenicol acetyltransferase (CAT), beta- galactosidase (beta-Gal) or beta-glucuronidase (GUS) have become popular in recent years. However, these co-reporter combinations diminish the performance advantages of luciferase. For example, while the luciferase assay can be performed and quantitated in seconds, the CAT, beta-Gal and GUS assays are endpoint assays requiring lengthy incubation periods prior to quantitation. Furthermore, these other reporters are limited in their sensitivity and in the range of their linear response; care must be taken not to exceed these ranges. Endogenous cellular activities can also interfere with the use of these reporters. Many cell types exhibit endogenous beta-Gal or GUS expression which hinder accurate quantitation of reporter gene expression, and endogenous deacetylase activity which interferes with assays of CAT activity. Although prior treatment of cell lysates at high temperature (1,2) can reduce the interfering endogenous activities for the beta-Gal and CAT assays, such treatment rapidly inactivates luciferase. Therefore, in dual-reporter measurements, this necessitates division and differential processing of co-transfected cell lysates prior to performing the reporter assays. The ideal dual-reporter method would allow the user to assay both reporters in a single sample with the speed, sensitivity, and linearity achievable with firefly luciferase. This is impossible with traditional reporters like CAT, beta-Gal and GUS due to limitations inherent in their assay chemistries and handling requirements. In contrast, Promega's Dual-Luciferase TM Reporter (DLR) Assay System meets these demanding criteria by combining the assays of two luciferase reporter systems, those of the firefly (Photinus pyralis) and the sea pansy (Renilla reniformis), in an integrated, single-tube assay format. Dual-Luciferase TM Reporter Assay chemistry Although both the firefly and Renilla luciferases provide the superior assay characteristics of bioluminescent reporters, they possess distinct evolutionary origins and, therefore, have dissimilar enzyme structures and substrate requirements. These differences have allowed us to develop the DLR Assay chemistry that selectively discriminates between these two luminescent reporter activities. Firefly luciferase is a 61kDa monomeric protein that does not require post-translational processing for enzymatic activity (3,4). Thus, it functions as a genetic reporter immediately upon translation. Photon emission occurs via oxidation of beetle luciferin in a reaction that requires ATP, Mg 2+ and O 2 (Figure 1 ). Under conventional reaction conditions, the oxidation of luciferin occurs through a luciferyl-