C-MYC AND CYCLIN D1 / RT-PCR - (May/22/2002 )
I saw a decrease in c-myc and cyclin D1 proteins expression level in WB (related to control) . But when trying to see the effect in RNA level using RT-PCR method I can not see any changes in the expression, and I don’t know why.
I know that the inhibitory affect I see is in the transcripional level (and not post translation).
Thank you for your help
The differences in mRNA probably are not so big to be detectable by a plain RT-PCR that even at 20 cycles can go to saturation . If your protein level is clearly down- regulated why do you want to try RT-PCR? I would not worry, but if you really have plenty of spare time try a semiquantitative RT-PCR or TaqMan PCR quantification.
as said before, your PCR maybe saturated. So try to determine a earlier stage were it will not be. (u can try ie: 20, 25 and 30 cycles or less... it depends on the srenght of expression.)