Quantify RNAi(siRNA) efficacy - Designing primers for quantification (May/18/2009 )
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?
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.
I don't think there is partical degradation. During RNAi, following the initial RISC cleavage of the mRNA, the whole mRNA is then degradated.