Firefly luciferase assays have made the quantification of genetic regulatory elements simple. By transfecting eukaryotic cells with an expression vector carrying the luciferase gene under control of a specific promoter, it is possible to monitor transcriptional activation of this promoter. The assay is based on the ability of luciferase, not normally expressed by eukaryotic cells, to generate light that can be quantified using a luminometer. This assay is combined with quantification of expression of a different reporter which allows a) normalization for transfection efficiency and b) differentiation between specific and nonspecific responses. Simplicity of use, sensitivity and reliability make this approach one of the most widely used methods to study transcriptional regulation.
The Promega Luciferase Assay System is a simple kit which combines a lysis buffer, used to obtain cell lysates, and a reaction substrate for the exogenously expressed luciferase. The assay itself is very easy once you have the construct containing the luciferase gene driven by the right promoter, thanks to ready-to-go reagents. Recently, we have been transfecting muscle cells with p21-Luciferase to monitor p53 transcriptional activity in these cells. We obtained cell lysates by scraping the cells cultures from 35 mm Petri dishes in 50 ul of Lysis Buffer. We then incubated aliquots of cell lysate with a pre-reconstituted solution of Luciferase substratum (stored at –20 ºC). After a few minutes of incubation, we aliquoted the mix into replicate tubes suitable for the luminometer. We recommend performing the readings in duplicate, since the variability can be high even at this step.
It is essential to normalize the experimental readings by a factor that accounts for the variability in transfection efficiency and gene expression in different cell cultures. Our main concern was to develop an alternative to the Promega Dual-Glo™ Luciferase Assay System (not available to us) which would have otherwise been the simplest and fastest solution for our needs (see related review). We decided to use the Promega Luciferase Assay System in combination with a fluorometric assay for Red Fluorescent Protein (dsRED) expression. The latter was driven by a strong, constitutively expressed promoter and its expression was therefore independent from the luciferase expression when co-transfected into our cell cultures. We found that the Lysis Buffer used for the luciferase assay did not interfere with dsRED excitation or fluorescence at 558 and 583 nm, respectively. Therefore, we first performed the luciferase assay, then we used an aliquot of the lysates to fluorometrically quantify their red fluorescence. The fluorometric readings were used to normalize the corresponding luminometric data.
This application of the Promega Luciferase Assay System, which is based on the expression of the firefly luciferase gene in combination with the dsRED, exploits a fluorometric approach to normalize the data. This combinatorial approach is more time consuming than a straightforward approach but is cheaper and equally reliable.
Research Associate
Department of Histology and Medical Embryology
University of Rome, La Sapienza