Recently, one of my fellow bloggers described some of the advantages of using dual-reporter assays (including our Dual-Luciferase®, Dual-Glo® Luciferase and our new NanoDLR™ assay debuting soon). These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.
While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:
Optimize the Transfection Efficiency
Every cell line is a little bit different. Some cells are easy to transfect and others much more difficult, requiring different reagents or techniques. Getting good transfection efficiency is key in getting high expression from a reporter assay. Spend some time to determine the optimal ratio of transfection reagent to DNA, as well as the total amount of DNA to transfect for your cell line. Don’t expect it to be exactly the same as that other cell line you’re working with.
One of the easiest ways to do this is to use a reporter vector (likely the vector you plan to use to normalize your dual-reporter assay) and test different conditions. Key to these experiments is to also include a mock-transfection control that will give you a sense of what kind of background you see in your cell line. You need to get good expression of your reporter, well-above background.
How do you know what’s above background? A common method is to take the average of your mock transfected cells signal (background) and calculate the standard deviation. Add three standard deviations of your mock transfected cell signal to your average background signal, and you’ve determined the minimal signal-above-background mark. In practice, you should see signal well-above background in transfection experiments where the the transfection efficiency is high.
Optimize the primary and secondary vector ratios
Just as important as the transfection efficiency is the ratio of primary to secondary reporter vectors. In practice, most people choose a secondary reporter driven by a viral promoter. CMV, SV40, and TK promoters are the general promoters of choice. CMV tends to be the strongest of these 3, followed by SV40 and TK.
While viral promoters are convenient to use, you should remember that their high level of expression means that you don’t need to add as much of the secondary reporter vector as the primary to observe high expression levels. And in fact, it’s almost never a good idea to do so. Remember that there is a limit to the amount of endogenous cellular machinery available to transcribe and translate reporter genes to mRNA to protein. If too much of that machinery is working to transcribe and translate the secondary reporter, you may in fact decrease the expression level of the primary reporter.
With that in mind, a really simple but useful experiment is to test different ratios of the primary to secondary vectors. Ratios of 10:1 to 50:1 primary:secondary might be good starting points, but don’t be surprised if you need to use 100:1 or 200:1 if you use a really strong promoter in your secondary vector. A general rule-of-thumb is that the signal of the secondary reporter needs to be just above background. More details of these experiments can be found in the following very useful article: Normalizing Genetic Reporter Assays Approaches and Considerations for Increasing Consistency and Statistical Significance.
Use a weak promoter for the secondary reporter
As explained above, strong promoters used in reporter vectors to normalize a primary reporter can impact the assay. For this reason, we often recommend a weaker viral promoter, TK. If you still get too high a level of expression in your cell line with the weakest promoters, you can try a promoterless vector. Just remember that the signal needs to be above background.
With all these points in mind, you should be well on your way to studying your gene of interest. One final tip is while all of these factors are important to get a good dual-reporter assay working, you may want to avoid the temptation to complicate your experiments by optimizing everything at once. If you work methodically to optimize each step individually, you should be gathering good data in no time.