Gene knock down via siRNA is often validated with their decrease in protein level (via Western Blot). Using qPCR, theoretically, it is not advisable since the primer can still amplify the region that are not targeted by the siRNA, meaning that region is still intact. Unless, we design a specific primer spanning the siRNA target area, which might result in no/reduced amplification. However, I once observed successful qPCR reading (less compared to the untreated sample) that shows significant gene knock down after quantification analysis. The primers used are not spanning any siRNA target area. However, [5'-siRNA1-----320bp-----Fwd. primer(100bp product)Rev. Primer-----200bp-----siRNA2-3']. There are approximately 650bp region between the siRNA1 and siRNA2, in which the primer amplify the 100bp region between them. So, can qPCR actually amplify a product from a ~700bp DNA strand (obtained from total RNA converted to cDNA)? And, does this means that qPCR can be used to evaluate knock down efficiency of a gene with high reliability and don't have to be reevaluated with Western Blot? I know it is theoretically unreliable but the results I seen make be a little bit suspicious now.
Hello Ahmed,
As the qPCR is base on RNA expression while siRNA are targeting RNA, but there is a way to check the full efficiency of siRNA by using Rifampicin with certain dilution to halt the transcription for a time being and then extract RNA during halt. so u may get the exact value for gene knock down.
As we were working with bacteria using ncRNA and after introducting of ncRNA expression cassette we used Rifampicin that worked properly.
Hi Ahmed,
we have published on degradation kinetics of siRNA targeted mRNA some years ago (https://doi.org/10.1016/j.bioeng.2004.09.001) Using different qPCR systems that have been designed to be flanking the siRNA binding site or either 3' or 5' of this site, we found that there might be slight differences in relative mRNA levels between 4h and 8h after transfection, but there's virtually no difference at 24h or 48h after transfection. This was true for high copy transcripts (ß-actin) and transcripts different in size (2.4kb to 5.7kb). So, our take home message was that the location of the PCR primers relative to the siRNA binding site is largely uncritical for the qPCR based determination of mRNA knockdown levels when analyzed not earlier than 24h post transfection.
Best, Peter
the moment mRNA is cleaved, it degrades rapidly. For most of mRNAs, it does not matter where TaqMan assay is located- upstream or downstream of siRNA target site, near or far. so qRT-PCR works quite well. Designing a TaqMan assay overlapping siRNA target site could be dangerous though. Few years ago it was thought that this is the best approach. However we observed instances with in vivo experiments, where there was no actual siRNA-mediated mRNA cleavage, but there was interference with reverse transcription (siRNA-derived oligos were co-purifying with mRNA) that negatively impacted subsequent qPCR and looked like a substantial knockdown
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