This may seem a naive question (and indeed it is); I'm in the process of using the dual luciferase (firefly and renilla) to quantitate the activity of my promoter of interest.
My question is: having established that between different transfections, the expression of my plasmid remain (more or less) constant, must I keep using the renilla reporter or could I skip it?
In other words, is the transfection of the renilla reporter always necessary?
Renilla transfection is necessary because it will discard manipulation mistakes.
Think that you put less volume in a well, renilla will normalize that mistake and you will not discard that datum because the ratio is what you evaluate. If you want to save money you could use some b-gal plasmid for data normalization it is a cheaper analysis but it is time consuming and also you have pipetting mistakes not discarded.
If you use protein levels it will not evaluate transfection efficiency or manipulation mistakes, but it is still used in some experiments.
If you will evaluate only the activity of a promoter in one cell line it is easy but in case that you use more than one cell line and different treatments, I recomend renilla normalization.
I think yes,it always necessary .because one advantage of this particular M2H system is the pBIND vector expresses Renilla luciferase, which corrects for deviations in transfection efficiencies (for example for pipetting errors, incorporation of DNA into cells, etc. ).
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