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[PaigeD]

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
or
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!

Thanks!

[pcrman]

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.