shRNA shows knockdown in protein expression but no change in RT-PCR - (Oct/04/2009 )
wondering if any kind einsteins out there can help me out. We have recently infected our cells with shRNA. Our western results show that the expression of the particular protein has been knockdown. However, to our surprise when we did RT-PCR, we see a strong band with intensity similar to our control. Does anyone have experienced something similar? The gene we are silencing is ATM. Thanks in advance.
Two potential explanations:
Explanation one is translational repression. Translation of the protein is being blocked but the mRNA levels are not going down... More akin to how miRNAs function
Explanation two is that you saturated your RT-PCR reaction.
Are you doing qRT-PCR in addition? For your RT-PCR, did you do serial dilutions of your input cDNA? It is really really easy to saturate a PCR reaction!
It sounds like you were doing conventional RT-PCR instead of realtime RT-PCR to detect mRNA degradation.
As MiBunny suggested, you might want to dilute your RNA. You might also want to design primers on two separated exons. I don't know much about ATM, any superfamily sharing the similar mRNA sequences?