Luciferase Assay Troubleshooting - Variable RLU from Renilla (Mar/13/2009 )
I'm doing a dual-luciferase assay with a promoter-firefly-reporter and a renilla-reporter control. I'm testing whether my protein of interest a transcription factor (which is cotransfected in) can activate the transcription from the promoter-firefly-reporter. I transfect HEK193 cells in triplicate.
Promoter-firefly-reporter + renilla-reporter + stuffer DNA
Promoter-firefly-reporter + renilla-reporter + Gene of interest
The DNA amounts stay constant. When I do the luciferase assay, I haven't been getting astounding results. The relative ratios are all pretty close. My PI ignored the ratios and noticed that the expression levels for the renilla-reporter decreases in the cells transfected with my gene of interest. The theory is that since my protein of interest is a transcription factor then it's interfering with the expression of Renilla.
Is there a way to control for this? How do I fix this? The renilla reporters I've tried are pRL-TK and pRL-SV40. Am I going to have to switch to a promoterless-renilla-reporter and is there one you recommend?
Right now my PI wants me to change cell types to see if that helps. Any advice would be greatly appreciated!
This occurs very often. The expression of the gene of interest may interfere the expression of TK reporter by a direct interaction or by affecting cell growth. In this case, you should try other internal controls such as beta-gal.