I designed few siRNA targeting a miRNA.
Northern blot shows that my siRNA degrades almost all of target miRNA.
I expected that degradation of miRNA would enhance the expression of a protein inhibited by this miRNA.
However, when I do a western blot for the protein, the expression level is very low with or without the siRNA treatment.
It seems like siRNA is lowering the miRNA amount significantly but not altering its function.
How can this be? Any suggestion on where to go from here?
Did you use siRNA to target miRNA precursor or "mature' miRNA?
Did you detect "mature" miRNA in the Northern blot?
Did you detect "mature" miRNA in the Northern blot?
QUOTE (sihyunie @ Oct 9 2009, 07:25 PM) 
I designed few siRNA targeting a miRNA.
Northern blot shows that my siRNA degrades almost all of target miRNA.
I expected that degradation of miRNA would enhance the expression of a protein inhibited by this miRNA.
However, when I do a western blot for the protein, the expression level is very low with or without the siRNA treatment.
It seems like siRNA is lowering the miRNA amount significantly but not altering its function.
How can this be? Any suggestion on where to go from here?
Northern blot shows that my siRNA degrades almost all of target miRNA.
I expected that degradation of miRNA would enhance the expression of a protein inhibited by this miRNA.
However, when I do a western blot for the protein, the expression level is very low with or without the siRNA treatment.
It seems like siRNA is lowering the miRNA amount significantly but not altering its function.
How can this be? Any suggestion on where to go from here?
Are u sure that the protein u tested is a real target of your miRNA? I mean: is it a predicted target or a validated one?
why choosing a sirna against a miRNA? an antisense approach sound more conventional and straightforward?
I'd try overexpressing the miRNA of interest to confirm that the protein you tested as a real target and then try the antisense approach against your miRNA.
hope it helps
Fizban
Another potential problem is redundancy. Your target gene is suppressed by multiple miRNAs, knockdown one of them won't release the mRNA from repression.
Thank you all for your response.
I believe that the protein target is a real target (it was given to me by my superior), but I tried using reporter systems with miRNA binding sequence (psiCHECK and such), and that didn't work as well.
I understand that antisense is more conventional method, but for now, we are stuck with using siRNA.
Another thought is that it might be a mechanical issue. siRNA in RISC can't bind to miRNA that's already in RISC.
I don't think it's the redundancy issue since antisense against miRNA recovers the protein level.
I believe that the protein target is a real target (it was given to me by my superior), but I tried using reporter systems with miRNA binding sequence (psiCHECK and such), and that didn't work as well.
I understand that antisense is more conventional method, but for now, we are stuck with using siRNA.
Another thought is that it might be a mechanical issue. siRNA in RISC can't bind to miRNA that's already in RISC.
I don't think it's the redundancy issue since antisense against miRNA recovers the protein level.
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