I've used Dharmacon SMARTpool siRNAs before but I've seen some articles which state that these low complexity pools (of 4 siRNAs) can lead to false positives/negatives. What do people consider the best approach?
Thanks
Dylan
Dear Dylan,
In the past we have used a second, non-overlapping siRNA set (Mission siRNA, Sigma Aldrich) to validate our findings with Dharmacon SMARTpool. To mimic the Dharmacon pool, we used two separate Mission siRNA's. After individually testing both, we combined them and repeated all relevant analyses (e.g. confocal, qPCR). We also re-validated our expression patterns with a non-targeting universal negative control. To close off, we added a BLAST analysis for our siRNA sequences, showing no off-target effects within relevant E-values.
Good luck,
Merle
Dear Dylan,
In the past we have used a second, non-overlapping siRNA set (Mission siRNA, Sigma Aldrich) to validate our findings with Dharmacon SMARTpool. To mimic the Dharmacon pool, we used two separate Mission siRNA's. After individually testing both, we combined them and repeated all relevant analyses (e.g. confocal, qPCR). We also re-validated our expression patterns with a non-targeting universal negative control. To close off, we added a BLAST analysis for our siRNA sequences, showing no off-target effects within relevant E-values.
Good luck,
Merle
SMARTpool proved to be no better than a single siRNA when it comes to off target effects.
Dear Dylan
Usually we start with OnTarget plus pool (with 4 si against the mRNA) and then we repeat the experiment with the individulas alone.
And the ultimate experiment is that you design your own siRNA against the UTr of the target and you rescue it with siRNA resistant plasmind (eg the cDNA)
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