A New Dual Luciferase Assay using NanoLuc Enables a Second-Generation Coincidence Reporter System to Reduce False Hits in HTS

The Dual-Luciferase® Reporter (DLR™) Assay System(a–f) provides an efficient means of performing two reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis or sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a luminescent signal lasting at least one minute. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated simultaneously by adding Stop & Glo® Reagent to the same sample. Both assays can be completed in about 4 seconds using a luminometer with reagent auto-injectors. In the DLR™ Assay System, both reporters yield linear assays with attomole (<10−18) sensitivities and no endogenous activity in the experimental host cells. Furthermore, the integrated format of the DLR™ Assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions. The pGL4 and phRL series of synthetic Renilla Luciferase Reporter Vectors are designed for use with the DLR™ Assay Systems. A Renilla luciferase vector with constitutive expression may be used in combination with any experimental firefly luciferase vector to co-transfect mammalian cells.