I want to detect if a gene's expression is reactivated when I knockdown the expression of my target gene but i'm not sure what sample to use for my standard curve. Basically, I have RNA samples from cancer cells which i have transfected with an siRNA to knockdown the expression of my gene A. I have done RT-PCR and confirmed that this has been successful & i have 80-90% suppression. My hypothesis is that when gene A is knocked down, gene B will start to be expressed again (as gene A represses gene B ). But how do I confirm this by RT-PCR? I used my non-targeting siRNA samples as my standard curve to check for the suppression of gene A but I cant use these samples for the standard curve of gene B as it wont be expressed due to the fact that gene A is repressing it. I have RNA samples for 24, 48 & 72 hours post transfection all of which have 80-90% suppression of gene A. Could I use one of these for my standard curve?
I'm sorry if this is a bit confusing.
Thanks in advance for any help.
Edited by moorele, 06 March 2009 - 04:44 AM.