renilla Luciferase and protein concentration - (Dec/02/2005 )

I am using the double Luciferase system to measure the a promoter activity in mammalian transfected cells. The Renilla Luciferase under the controll of TK promoter is used to normalize the activity of the promoter of interest. Is it right to normalize also the renilla luciferase by the amount of total protein ? I don't belive so because the Renilla values are not proportional to the amount of protein or to the amount of cell since in every transfection the copy of plasmid that can get into a cells is never equal to 1. Do you think I am right or not?

I have been doing transfections (amongst other things) for several years now. The last couple of years I have been using the Renilla from promega as a transfection control and like many others saw odd things when I began adding certain transcription factors. One thing which worked for me was changing to pHRL, though even with this system things can sometime appear strange.

As I am one who firmly believes that mathmatics is good for analyzing your data, but should absolutely not be used for trying to change data to derive values which are more what you would like to see (this should b a golden rule in any lab!), maybe we should try and think about what we are observing. We mustnt forget we are still working in an environment that we dont yet fully understand. Many transcription factors might not just simply be having an effect on our promoter of interest, but on the cells transcriptome as a whole. Is it too wild to think that certain transcription factors, although inducing our promoters of interest... may have effects on the cell which make it a less suitable environment for other promoters ? Lets face it, if its black and white why do some cell lines become harder to transfect as they age , where as others seem to remain happy for many generations ?

More pragmatic solutions would be:
Use other cell-lines
Look at your cells before and after transfection
Use other reporters (not just Renilla, beta-gal, but try using GFP)
If you know where your protein binds, make a mutant
Mutate your protein's DNA binding site (often literature provides examples of already found mutations)
What kiind of things does the protein do (downstream effects, eg. cell-cycle)
Add small amounts and do titrations (anyone can see an effect if you feed a cell ugs of TFs, try ngs and do a full range of titrations)

I am curious to know how other people approach such problems....


Phil