2 replies to this topic

[vickie]

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

vickie

[Elzbieta Janda]

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.  

[scogne]

Hi,

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.)
Good luck.