Crosstalk between luciferase and GFP in Dual Luciferase Assay (Promega)?
Posted 19 April 2011 - 11:59 AM
do you know anything about crosstalk of firefly luciferase with GFP and renilla luciferase with GFP?
I did transfections of different cell types, with a pGL3 vector, which carries a native promoter (nkx 2.5 promoter)- from this vector the firefly luciferase is expressed. To correct deviation from transfection efficiency the renilla luciferase vector phRL was co-transfected (1:10). A third plasmid type was co-transfected into some of the samples: it was a short hairpin RNA-GFP expressing vector (for transient knockdown at mRNA level)- I got really strange results from the combination with GFP. Is it possible that luciferase crosstalks to GFP? does this rather increase or reduce my signal from luciferases or nothing, because the "amount" of photons, which are detected in the luminometer, stays the same??
Posted 19 April 2011 - 02:01 PM
We have tested GFP expression and its impact on the signal obtained by Dual-Luciferase assays and have not found it to be an issue. What could be happening is that the expression from the shRNA vector changes the transcriptional dynamics of the luciferases. I would recommned testing a control of your shRNA expression vector without an insert to eliminate the possibility that the shRNA itself impacts luciferase expression. I would also recommend getting in contact with Promega Technical Services as they will be able to help review your data and make further recommendations.
Edited by KevinK, 19 April 2011 - 02:02 PM.