Luciferase assays are widely used to quantify gene expression. Typically, they are used following cells transfection with an expression vector carrying the luciferase gene under control of a specific promoter of interest. By quantifying the amount of light produced by the luciferase-based enzymatic reaction, it is possible to monitor transcriptional activation of this promoter. Quantification is performed by using a luminometer. This assay must be combined with quantification of the gene expression of a different reporter which allows for a) normalization for transfection efficiency and, b) differentiation between specific and nonspecific responses.
The Promega Dual-Glo™ Luciferase Assay System is a kit which offers the possibility of quantifying both firefly and Renilla gene expression in mammalian cells in a few steps and in sequence. With this approach, the two luciferase activities can be exploited to monitor reporter gene activity and normalize gene activity in a single sample. The two luciferase activities elicited by two different reagents (combined with a quenching process) allow specificity of signal. The presence of stabilizers for the luminescent signals hampers the rapid decay of the signal, thus allowing quantification of a large number of samples even in manual operating mode. A very convenient feature of the Dual-Glo™ Reagent (see below) is that it can be subjected to several cycles of thawing and freezing without affecting the output of the assay: This avoids waste of very expensive reagents. The manufacturer sells vectors bearing the firefly or Renilla luciferase genes, which can be used to subclone the promoter of interest so to have it driving the luciferase expression.
In my former laboratory, I used this system to monitor the transcriptional activity of a muscle gene exogenously expressed in fibroblasts upon different cell culture conditions. I typically co-expressed in the same cell population: 1) the muscle gene of interest driven by the SV40 promoter; 2) a target gene promoter subcloned in the firefly luciferase construct; 3) the Renilla luciferase driven by the constitutively active HSV-promoter. After one or two days of cell culture I obtained the results in about 1-2 hrs (depending on the number of samples). I followed the simple 5 step protocol which follows: 1) I prepared the Dual-Glo™ Reagent by mixing the Luciferase Buffer with the Luciferase Substrate; 2) I incubated the cell culture with the reagent for 15 min; 3) I transferred the samples to luminometer tubes and performed the readings; 4) I added the Stop-and-Go Reagent to the tubes and incubated for 15 min; 5) I repeated the measurements in the luminometer. The first measurement (firefly) was normalized by the second (Renilla) and replicate samples were averaged.
The protocol seems very standardized: It is almost impossible to do something wrong. The high sensitivity of the system allows miniaturization. Given these key features, I had a positive experience with the kit. When I found myself in hard times because of cost limits, I adopted an alternative approach using the Promega Luciferase Assay Kit instead.
Research Associate
Department of Histology and Medical Embryology
University of Rome, La Sapienza