Genetic reporters such as firefly luciferase are commonly used in molecular and cellular biology research to study a myriad of different things including gene expression, promoter activity, intracellular signal transduction, apoptosis, mRNA processing, and protein:protein interactions. I used Promega’s Dual-Luciferase® Reporter Assay System to study a variety of promoters that respond to Notch signaling. The kit has been developed for reporter quantification in mammalian cells and includes a proprietary Cell Culture Lysis Reagent which must be used.
The Dual-Luciferase Assay System works on the principle of using different enzymes found in the luciferase family that use different substrates. These enzymes are called firefly luciferase and renilla luciferase. These enzymes interact with their substrate and upon doing so generate a product as well as light which is measured by an instrument (the luminometer which is described below). Promega’s instruction manual provides an excellent description, as well as a descriptive cartoon, to see the chemistry involved. Essentially, you lyse your cells to create an extract (must use a Promega lysis buffer, the kit comes with Passive Lysis Buffer which works very nicely), add the firefly reagent (called LARII) first and take a measurement to quantify the amount of firefly luciferase present, then add the renilla luciferase reagent (called Stop&Glo) and take a measurement to quantify the amount of renilla luciferase present. The renilla luciferase reagent contains a proprietary agent which quenches both the light and firefly enzyme immediately from the first reaction. Therefore, when your instrument takes the reading, the light is proportional the enzyme being measured. A word of caution though, do not accidently add the renilla luciferase reagent first as the reagents completely inactivates the firefly luciferase enzyme. You must go in order from firefly to renilla substrate every time.
This kit contains Luciferase Assay Reagent in lyophilized form; before use, the reagent must be mixed with provided buffer (LAR II). The reconstituted LARII mixture can be stored at -80°C for up to 6 months. I recommend storing in 1 ml aliquots. The Renilla reagent must be prepared fresh each time although it can be temporarily stored at -80°C for up to one week. Both reagents are light sensitive so keep covered until ready for use.
Remove an aliquot based upon the amount needed and allow it to come to room temperature before use. Usually, 100 µl of Luciferase Assay Reagent is sufficient for the reaction. I have found that you can actually quantify this and use less reagent. For example, we use 25ul of reagent for 96-well plate format thereby maximizing the reagent use. As a rule, use equal amounts of reagent with cell lysate. The light emitting reaction happens when you mix your sample with Luciferase Assay Reagent. Maximum light intensity is stable for at least 1 minute and begins to decrease afterwards. Therefore, your readings must be taken fairly quickly after addition of the appropriate substrate. I have found it ideal to take a measurement within 5 seconds of addition of the substrate. I have also found that it is possible to lyse cells and freeze them in the Passive Lysis Buffer at -20°C or -80°C until you are ready to take the measurement.
Different types of luminometers can be used with this system. I use both a single-tube luminometer (Turner Design Modulus) and a 96-well plate luminometer with dual injectors (Turner Design Veritas). The instruments are very sensitive so time must be taken to determine the amount of cell extract and the amount of substrate needed for the reaction. However, in my hands, we use HeLa and U2OS cells in a 96-well format, lyse in 25 ul of Passive Lysis Buffer, and use 25 ul of firefly substrate followed by 25 ul of renilla substrate and get consistent results.
As a conclusion, I can say that this assay is easy and relatively quick to perform; it gives very reliable and reproducible results. I highly recommend it.