pGL3 luc reporter assay - controls needed (Jan/08/2008 )
Hi,
I' d like to know how many controls do I have to use when doing an activity assay with luciferase..
Renilla plasmid what kind of control is?
thanks
biotech
Co-transfection with renilla luciferase normalizes for differences in transfection efficiecncy, assuming you can do both firefly reporter (like pGL3-luc) and a renilla reporter driven by a 'housekeeping' system (eg pRL-TK driven by the thymidylate kinase promoter). You can (in my opinion, should) also normalize for well-to-well variability in cell number & cytotoxicity induced during the transfection process, and can be accomplished by any number of methods. You could also perform transfections with a non-reporter plasmid, such as pGL3 without your promoter to subtract the any cross-activation (should be zero) and controls with only firefly or only renilla luciferase. -JAH
oK,
how many controls, so let me see if I get it...
I want to test a promoter activity, I cloned it on pGL3 plasmid..
then when testing activity I have to do the following:
I co transfect with Renilla plasmid...as a constant value to normalize activity,
then I have to find a method to also test that I have the same amount of cells in each sample.....
the promoter less pGL3 is another independent sample, right? I mean
sample 1: promoter less and Renilla.....
2: pGL3 promoter 1 + renilla
3: pGl3 promoter 2 + renilla
etc, etc
each with different length promoter fragments..
Finally, in different papers I've read different strategies...
some use pGL3 and Renilla
others pGL3 and b-gal
and some also used pGL3 and proteins concentration (Bradfore) as control..
Renilla is the best?
thanks for your help!
biotech