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Quantify RNAi(siRNA) efficacy


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#1 dcch

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Posted 18 May 2009 - 06:41 PM

Hello.

I am currently in my experimental design on RNAi work. In one of the steps, i would like to measure RNAi efficacy by using qRT-PCR method. Do i need to consider the amplification region in cDNA? It is because i am worried if the amplification region is outside of RISC cleavage site, the partially degraded mRNA (after RISC cleavage) will interfere my results.

Or this is something that i think too much, where partially degraded mRNA is insignificant and neglectable, or even absent in nature?

Thank you.

Edited by dcch, 18 May 2009 - 06:44 PM.


#2 miRNA man

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Posted 19 May 2009 - 10:58 AM

If your siRNA isn't perfectly complementary (like a miRNA) then partial mRNA degradation or translational inhibition is a real possibility. You could check the protein level instead, which might give a clearer picture if you are concerned about partial degradation.

#3 pcrman

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Posted 19 May 2009 - 12:12 PM

I don't think there is partical degradation. During RNAi, following the initial RISC cleavage of the mRNA, the whole mRNA is then degradated.




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