using EGFP fluorescence as measure of transfection efficiency - (Apr/02/2007 )
I am currently doing promoter experiments where I transfect a plasmid containing a reporter gene and on the other arm of the construct, a CMV driven EGFP gene. After transfection, I measure EGFP fluorescence with a spectrophotometer multiwell scan ( spectramax) as an indicator of transfection efficiency.
The fluorescence values I get for my control and untransfected wells are similar or slightly less than my sample wells. How does this occur? I also notice that if I adjust the max RFU values, I can obtain different readings for the same well! Am really very confused.
Would appreciate input
Do not use plate reader for EGFP. It is not sensitive enough. FACS analysis would be a better choice if you want to stick with EGFP. EGFP is too stable for promoter studies. That is another reason that you'd be better off to switch to other systems. You alternatives are luciferase and b-Gal.
thanks for your reply. I am using beta gal as the reporter gene whilst EGFP is used to normalise for transfection efficiency ( both beta gal and EGFP are within the same construct)
Pardon me for sounding like an idiot but what do you mean by EGFP is too stable for promoter studies
Have a nice day
Yes. A protein with short halflife will present you with a more acurate, dynamic level of the reporter gene product. A stable protein gives you a cumulative level of this protein over time, which remains high even when the promoter is turned off.
Reporter with luciferase and b-Gal as nromalization will be a better combination.