siRNA off-target effects can be reduced at concentrations that match their individual potency

This study demonstrates that siRNA off-targets (e.g. 3'UTR off-targets), can be significantly reduced when cells are treated with a relatively low dose of siRNA (e.g. 1nM) that is sufficient to effectively silence the intended target. Cells were treated with different concentrations of either unmodified siRNA or modified siRNA that targeted either hexokinase2 or STAT3 (HK2-3581, HK2-4031, HK2-3581M, STAT3-1676, STAT3-1676M). As transfection efficiencies can vary according to the ratio of nucleotide to transfection reagent, each dose was supplemented with non-targeting/negative control siRNA (siGenome2 or Allstars negative control), such that the total RNA concentration was equal across experiments. RNA was purified 48 hours post-transfection using the RNeasy mini kit from Qiagen. Biological replicates were normalized using the RMA method and analyzed using various Bioconductor tools.