Reporter genes are widely used for the rapid evaluation of cellular physiology. The process is usually quite simple; just transiently transfect the reporter vector into your cells, then analyze the reporter gene’s expression after 24 or 48 hours. It offers an excellent means for studying complex genetic regulatory networks. But, during the experiment, there can be a lot of variability: differences in cell seeding numbers, transfection efficiency, pipetting volumes, cell lysis efficiency, assay efficiency and cell viability - cytotoxicity can be confused with down-regulation of reporter expression.
Promega’s Dual Luciferase Reporter Assay (DLR) overcomes many of these disadvantages by integrating the assays of two luciferase reporters: one as the experimental reporter, another as the normal control. During analysis, they are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding luciferase assay reagentII (LARII). The renilla luciferase reaction is initiated by adding the Stop&Glo reagent to quench the first reaction. The protocol is as follows: first, cells can be lysed in 1xPLB (supplied in the kit) at room-temperature for 15 minutes. The lysate is quite stable and can be kept at room temperature for several hours. We have even put lysates through several freeze/thaw cycles and have seen no big differences in reporter gene activity. After the cells have been lysed, the LARII and Stop&Glo reagents are prepared (you can get a little more of the Stop&Glo reagent by transferring 220ul substrate into the 11ml substrate solution). Once this is complete, it is just a matter of incubating the lysate and substrate and reading the results on a luminometer. The DLR assay is rapid - you can get numbers for both reporters within 30 seconds. If you use a single sample luminometer, using as little as 10ul of cell lysate is enough for a reading. In fact, sometimes the reading is too high (above 1 million RLU) and the machine will display an error message. When this happens, you simply have to cut down the amount of cell lysate used. If you use a multi-sample or plate reading luminometer, there are usually automatic controls for the injection of both reagents and the interval waiting time is consistently 2 seconds. The downside of these devices is that they will need about 2ml of each reagent to prime its tube system. In addition, we have had the reagent used up during the reading and injection steps. This results in extremely low (or blank) readings. Thus, if you are using a multi-plate luminometer, it is important to check the reagent level regularly during its use.
Although the internal control is a good idea, we have encountered some issues with this system. In particular, we have had problems with the control vector behaving strangely. In particular, there can be problems with it being sensitive to experimental stimulations, which can affect results. Because of this, Promega has developed 6 different control vectors. Thus it is important to find a stable control vector which does not respond to your experiment stimulation. Although Promega claimed that they removed most of the potential transcription factor binding sites from the phRL vector backbone and promoter, which contributes to the undesirable modulation of reporter vector expression, my experience with phRL-TK is that it still shows some sensitivity to stimulation. In our hands, the phRL-CMV works better. The bottom line is that it is important to choose the one that suit for you.
Xiang Yang, Ph.D.