Dear all,
I am working on an uncharacterised protein. The function of this protein in mammalian cell is yet to be discovered. I over-expressed the protein did interaction and co-localization studies. We tried to knock-down this protein using siRNA targeting a specific site, surprisingly we found that after siRNA transfection the mRNA level of this protein increased 5 fold (technically I should see a reduction in mRNA). Had anyone came across this type of result??.Is it possible for siRNA, targeting a specific region of a mRNA to increase mRNA level directly or indirectly??or else the increase in mRNA which I am observing could be a experimental error??
Hi,
An experimental mistake is always possible... But there might be other explanations!
Working with siRNA can be tricky. Things you can do:
*check your rt-PCR product on an agarose gel, if you get several bands maybe it's a primer problem explaining the 5-fold increase.
*As said before, check the protein level.
*change the transfection timing: I get good siRNA knock down around 48h post transfection (with up to 24h of siRNA treatment), 72h to see it on protein level.
*change the transfection method, try Lipofectamine RNAiMAX for example.
*try another siRNA if possible. It's good to try 2 different ones , they might have huge off-targets effects.
Finally, maybe this uncharacterised protein is so important that the cells have some kind of "protection mecanism" increasing its transcription when you try to knock it down...
I hope this helps!
Have you validated your reference gene to be stable in your mode/condition?
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