Figure 2. Promega's DLRTM Assay System.
In the Dual-Luciferase Reporter Assay System, firefly luciferase activity is used as the "test reporter" to monitor the biological activity of interest. For example, the firefly luciferase may be coupled to a regulated promoter to study the structural or physiological basis of regulated gene expression. Changes in the transcriptional activity of the promoter under study would result in relative changes in the expression, and thus measured activity, of the firefly luciferase reporter. The DLR System uses the luciferase from the sea pansy as a "control reporter" to establish an internal baseline by which each measurement can be normalized. To effect this, the sea pansy luciferase is coupled to a constitutive promoter that is unperturbed by the experimental conditions.
Both luciferases catalyze bioluminescent reactions. They work well together as co-reporters because they catalyze different reactions, yet are compatible in terms of handling conditions as well as reaction sensitivities and linear ranges. Firefly luciferase is a 61 kDa single-subunit enzyme that catalyzes the ATP-dependent oxidation of beetle luciferin. The Renilla luciferase is a 36 kDa monomeric enzyme that catalyzes the oxidation of coelenterazine. Under conventional reaction conditions, the oxidation of luciferin by the firefly enzyme results in a flash of light that rapidly decays. Promega's patented assay reagents utilize a coenzyme that alters the kinetics of this reaction and results in an extended luminescence signal that can be readily and reliably measured.
According to Promega, the linear range of its firefly luciferase assay extends over eight orders of magnitude, with a lower detection limit of about 10-20 mol of the reporter. Similarly, the assay for the sea pansy enzyme provides over seven logs of linearity, with a sensitivity of approximately 3 x 10-19 mol.
One of the advantages of Promega's DLR System is that the two bioluminescent assays are performed sequentially, in one tube. Dr. Harold Bernstein, Cardiovascular Research Institute, University of California, San Francisco, says , "My group uses Promega's Dual-LuciferaseTM Reporter Assay System because it avoids having to do a second assay (for example, (ß-galactosidase) for transfection efficiency." This is in contrast to other dual-reporter applications, which use firefly luciferase in combination with nonluciferase reporters (such as chloramphenicol acetyltransferase, ß-glucuronidase, or ß-galactosidase). The handling requirements, reaction kinetics, and/or assay methods for these traditional reporters are sufficiently different from those of the luciferases and require sample division for separate assays. It takes less than one minute to perform both luciferase assays in Promega's DLR System. In a luminometer equipped with reagent injectors, both assays may be completed in as little as 4 seconds.
First, the firefly luciferase is quantified after the addition of Luciferase Assay Reagent I (LAR II) to the sample. The sea pansy luciferase is then quantified after the addition of Stop & Glo® Reagent. This reagent quenches the firefly luminescence, and initiates the reaction catalyzed by the Renilla luciferase.