siRNA causing mRNA knockdown but no protein knockdown! - (Dec/15/2005 )

Hi,

I have encountered a phenomenon that I cannot resolve and am looking for some suggestions, or if anyone has encountered a similar situation:

I have designed 2 siRNAs against the same target gene. On transfection, I see 60% mRNA knockdown (at 24h), relative to missense siRNA-transfected cells. However I do not see any protein knockdown at 24,48 or 72 h post transfection for either siRNA. In addition I see a phenotypic effect for one of the 2 siRNAs, but no the other, even though there appears to be comparable knockdown at the mRNA level.

So, there appears to be 2 issues - 1. is the protein data or RNA data correct, and 2. is the phenotypic effect of one siRNA due to off target effects.

If anyone can shed any light, or empathse, I would be gratefull to hear from you.
KeK

Do you know the half-life of the protein you are trying to knock out?

Maybe the protein is not easily degraded.

Are there known pseudogenes for this molecule?

If the siRNAs are not specific for the true transcript, the pseudogenes could be mopping them up. This could also explain why only one gives a phenotypic response.

Did you also try and knock-out a housekeeping gene/ protein?

i.e. can you see a decrease in protein concentration related to a reduced mRNA level for other genes.

KeK,
I have been having the same problem. My siRNA knocks down mRNA levels wonderfully after 24hr, but my westerns do not show any knock-down, 48hr post. How were you able to resolve your problem? My boss mentioned determining the half-life of my protein, but I have not been able to find a protocol to determine protein half-life. Any help would be wonderful!
Thanks,
unccf

It turned out that the mRNA of our target was very unstable and so sometimes appeared to have a long half life and others, short. We have not continued with this target. Unfortunately I have no advice on how to measure the half life of a protein. Are you certain about the specificity of your antibody? May sound silly but have you tried loading different amounts of protein on your WB to validate that the antibody can pick up different amounts of protein - maybe it is an antibody problem. Are there other isoform of that target hat may not be reduced by the siRNA and not picked up by QPCR but are picked up by the antibody?
A time course : 24, 48 72 h for siRNA knockdown on WB and parallel QPCR might show up something.
Sorry there are more questions than answers!
KeK

Check out the half life of the protein. If the protein is stable, you might need upto a week to actually see a change in the protein levels.