Dual-Luciferase Reporter Assay System From Promega

The luciferase assay is one of the most well-known and convenient reporter assays. In particular, Promega has developed a firefly luciferase reporter system to study the regulation of gene expression in mammalian cell culture. To accomodate the variety of needs in the field of gene expression research, Promega provide several different vectors, such as the pGL3 Basic Vector which only has the luciferase gene and lacks regulatory regions, such as promoter and enhancer sequences. This allows for maximum flexibility in cloning putative regulatory sequences. The pGL3 Enhancer Vector contains an SV40 enhancer downstream of the luciferase gene and the poly(A) signal. This vector is useful in the study of functional promoter elements to regulate gene expression because transcription of the luciferase gene is maintained at a higher level by using the SV40 enhancer. The pGL3 Promoter Vector contains an SV40 promoter upstream of the luciferase gene. Possible enhancers can be verified by using this vector by inserting the enhancer either up or downstream of the promoter. Finally, the pGL3 Control Vector contains the SV40 promoter and enhancer sequences and therefore, it strongly expresses the luciferase gene in many types of mammalian cells. It can be used as a positive control for experiments for such things as monitoring the efficiency of transfection. Promega also provides Renilla luciferase constructs such as the pRL-TK and pRL-SV40 vectors to normalize and reduce differences in transfection efficiencies and subsequent variations in these experiments. The HSV-thymidine kinase promoter (pRL-TK) is a relatively weak promoter compared to the early SV40 enhancer/promoter (pRL-SV40). Therefore, the Renilla luciferase level for pRL-SV40 is almost 10 times higher than the Renilla luciferase level for pRL-TK from my experience. For this reason, the relative luciferase activity (RLU) level which was normalized by pRL-TK will show relatively high luciferase activity compared to pRL-SV40. The Dual-Luciferase® Reporter Assay System by Promega also provides Luciferase Assay Buffer II, Luciferase Assay Substrate (for firefly luciferase), Stop & Glo® Buffer, Stop & Glo® Substrate (for Renilla luciferase), and Passive Lysis Buffer.

The firefly luciferase reporter assay is initiated by adding a cell lysate to the Luciferase Assay Reagent II. After reading the output of the firefly luciferase by luminometer, quenching of the firefly luciferase luminescence and concomitant activation of Renilla luciferase are accomplished by adding Stop & Glo® Reagent (The Stop & Glo® substrate is supplied at a 50X concentration). To prepare working concentration, add 1 volume of 50X Stop & Glo® Substrate to 50 volumes of Stop & Glo® Buffer in a glass or siliconized polypropylene tube. After the adding Stop & Glo® Reagent, the output of the Renilla luciferase is then read by luminometer.

For our luciferase assays, approximately 5x104 of HeLa cells were seeded in a 24-well plate in antibiotic-free media. After attachment, cells were transfected with luciferase constructs (one of the pGL3 constructs for firefly luciferase and one of the pRL constructs for Renilla luciferase) by using Lipofectamine 2000 (Invitrogen). After 24 hr incubation with cell culture media, cells were lysed in passive lysis buffer (Promega). Firefly and Renilla luciferase signals were measured by the Dual-Luciferase® Reporter Assay System (Promega) in a Junior LB 9509 luminometer (Berthold Technologies).