Suppression seen at protein levels but not at RNA - Pls Help - (Feb/20/2008 )
Hi Guys,
I have created stable cell lines with siRNA, they are under the drug selection.
I am seen almost 80% reduction in protein levels, but there is no change at the RNA level.
Can I trust these results, I have repeated western atleast 4 times to confirm my results....
Has anyone encountered this problem???
Any suggestions with be helpful,
Thanks
Sounds to me like a miRNA-mediated knockdown of your protein. For miRNAs, many targets are silenced by translational repression, not degradation of the mRNA. Do you know the sequence of your siRNA? If so, check to see if it is perfectly complementary to the target, or if there is a bulge at ~9-11nt (if there is then cleavage of your target is probably unlikely and would indicate miRNA repression mechanism). Also be sure to check the 3' UTR since this is where the majority of miRNAs are thought to bind.
Thank you miRNA,
Unfortunately, I do not have the sequence to check whether it is complementary to my target sequence or not. My target is an enzyme, I am planning to check its enzymatic activity.
But, do you think, it is worth while using this sample to check for microarray studies, which is what I want to do ultimately.
Thanks a lot for your help,
Abheda
I'm not sure I understand your question - what would the goal of your microarray experiment be? To knockdown your protein with siRNA but to check secondary/indirect effects on other mRNAs?
I agree with miRNA man, we need more info on what are you doing. how have you designed the siRNA if you dont know its sequence or gene sequence it is targeting?
translational repression without degradation would be unusual for siRNA to my knowledge, but not impossible.
I agree with miRNA man, we need more info on what are you doing. how have you designed the siRNA if you dont know its sequence or gene sequence it is targeting?
translational repression without degradation would be unusual for siRNA to my knowledge, but not impossible.