renilla values are going down with firefly values after miRNA transfection - (Jun/23/2011 )
Im doing dual luciferase assay (Promega) for looking at 3'UTR activity and inhibition by miRNA. The vector has both firefly (under the control of PGK promoter) and renilla (under SV40 promoter). Now I have cloned this 3'UTR immediately after firefly luciferase in the MCS. I transfected the cells with the 3'UTR vector alone, 3'UTR vector+pre-miR . What I noticed was although the firefly values are going down with the mir transfection, renilla values go down as well. I used a negative control miR (synthetic sequence)where the firefly and renilla values stay pretty stable just like the vector alone. I understand that renilla values basically infer the efficiency of transfection but in this context the ideal condition should be when after miR transfection, renilla values more or less remain unaltered. Would like to hear out any suggestions on what controls should I keep and how to improve my experimental system.
It seems that if the negetive control is not showing this decrease in Renilla signal, the most likely scenario is that the miRNA is down regulating both the experimental firefly luciferase signal and the control. Have you looked at the Renilla sequence to see if there are any predicted binding sites for your miRNA of interest? You might also be able to generate a mutated binding site in your 3'-UTR of interest to show that the firefly signal is stable when the miRNA binding is eliminated.
I hope these recommendations are helpful and it would be useful to hear from others with experience in this field. Please do not hesitate to contact Promega Technical Services if you have any further questions or concerns. We have scientists across the world who are there to help with such questions.