This post was contributed by guest blogger, Scott Messenger, Technical Support Scientist 2 at Promega Corporation.
It’s always an exciting time in the lab when you find a new assay to answer an important research question. Once you get your hands on the assay, it is always good to confirm it will work for your experimental setup. Repeating the control experiment shown in the technical manual is a great way to test the assay in your hands.
After running that first experiment of your assay, it looks pretty good. The trends of control and treatment are consistent. Time to get on with the experiments…but wait—the RLUs (Relative Light Units) are two orders of magnitude lower than the example data! I can’t show this data to my colleagues; it doesn’t match. What did I do wrong?
This is a concern that we in Technical Services hear frequently. The concern is real, and I had this same thought when doing some of my first experiments using luminescence. When a question like this comes in, a Technical Service Scientist will make sure the experiment was performed as we described, and in most cases it is. We then start talking about RLUs (Relative Light Units).
Bioluminescent reporter assays are an excellent choice for analyzing gene regulation because they provide higher sensitivity, wider dynamic range and better signal-to-background ratios compared to colorimetric or fluorescent assays. In a typical genetic reporter assay, cells are transfected with a vector that contains the sequence of interest cloned upstream of a reporter gene, and the reporter activity is used to determine how the target sequence influences gene expression under experimental conditions. A second control reporter encoded on the same or a different plasmid is an essential internal control. The secondary reporter is used to normalize the data and compensate for variability caused by differences in cell number, lysis efficiency, cell viability, transfection efficiency, temperature, and measurement time.
For genetic reporter assays, using a secondary control vector with a weak promoter like PGK or TK to ensures that the control does not interfere with activation of your primary reporter vector. Transfection of high amounts of the control plasmid or putting the control reporter under control of a strong promoter like CMV or SV40 often leads to transcriptional squelching or other interference with the experimental promoter (i.e., trans effects). Reporter assays can also be used to quantitatively evaluate microRNA activity by inserting miRNA target sites downstream or 3´ of the reporter gene. For example, the pmirGLO Dual-Luciferase miRNA Target Expression Vector is based on dual-luciferase technology, with firefly luciferase as the primary reporter to monitor mRNA regulation and Renilla luciferase as a control reporter for normalization.
Here in Technical Services we often talk with researchers who are just starting their project and looking for advice on designing their genetic reporter vector. They have questions like:
How much of the upstream promoter region should be included in the vector?
How many copies of a response element will be needed to provide a good response?
Does the location of the element or surrounding sequence alter gene regulation?
Luciferase assays are useful tools for studying a wide range of biological questions. They can be performed easily by adding a reagent that provides components necessary to generate a luminescent signal directly to cells or a cell lysate. However, once this reagent has been added, how long you wait to measure the signal becomes a key consideration in generating consistent data. Dependent on which luciferase assay you use, you may need a luminometer that can use injectors to deliver the assay reagents. The reason for this is simple, but can be confusing to new users.