Typically, the effect of siRNAs are assayed after 48hrs. During this period, the siRNA is expected to "keep degrading" the existing as well as newly synthesized transcripts. Now, if there is a pool of target transcripts and siRNAs are transfected, how long would it take to have all existing transcripts degraded (considering no new target transcript is being made)? Is there any such mechanistic information available? Conversely, if cells are flooded with siRNAs and then a certain short-lived transcript (say, half-life of 2 hrs) is induced, would the "knocking down" be detectable by sensitive assays like GRO-seq within the same 2 hrs induction window? Any idea/thoughts/information will be highly appreciated.
Hi Anil,
The effect of siRNAs is very variable, depend on your cell-line, on your transcripts and the cell processus which will be involved in the degradation. I think that the best way is to do a kentic pre-study to fix when the degradation start and when it attempt the maximum of efficiency.
That's true, Amine, thanks. My question was on the molecular mechanism per se of RNA degradation. I should reframe the questions: (1) if targeting siRNA duplexes are flooding the cytoplasm, how long does it take, statistically, for the duplex to find a newly synthesized target--assuming that the target is being made in moderate abundance (again, I know "moderate abundance" is not a quantitative term; and you know what I mean)? (2) once the duplex finds the target, how long does it take to assemble the entire RISC complex and to degrade the target--assuming that the RISC is also of "moderate abundance" in the given cell type?
Hi Anil,
the effect of siRNAs depend on employed cell line, but you can usually observe siRNA effects after 48-72h.
Hi Erica,
Thanks for joining the discussion. But please review whatever has already been said before.
My question DOES NOT relate to how long it takes to detect gene knockdown (either transcript level by RTqPCR or protein level by Western); I know it depends greatly on several factors including the cell type, efficiency of transfection, efficacy and abundance of the siRNA duplex, stability and turnover of the concerned transcript, the kinetics of re-synthesis of the concerned transcript etc.
My questions are:
1. How long does it take for a population of siRNA duplexes in the cell to find the target transcript? Quantitative parameters here could be, say, 25-50 nM duplexes, and ~100 copies of the transcript per cell.
2. Once the duplex finds its target, how long does it take for assembling the RISC complex and to finally complete transcript destruction.
I shall appreciate if you could provide specific insights. Thanks again.
mRNA degradation happens very fast- if you have a potent siRNA, and efficient siRNA delivery into cells of interest, mRNA knockdown of >80% can be measured by qRT-PCR as early as 6 hours; it peaks at about 12h. at the protein level it takes longer to see the effects of course, many folks use 24-48h time point for assay, depending on the target etc
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