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Inducing RNAi with siRNA Cocktails Generated by RNase III

uggest that nonspecific gene silencing is not occurring in cells after transfection with RNase III generated siRNA cocktails. A recent article that examined the effect of RNase III generated siRNA cocktails on related RNA binding proteins confirms the lack of nonspecific effects (5). In non-mammalian systems, mixtures of siRNAs to multiple sites within the target gene are generated in vitro by Dicer digestion of long dsRNA, which in turn specifically silence target genes. This suggests that there are mechanisms in place to maintain a high degree of specificity.

Comparison of RNase III Generated siRNAs to Individual Chemically Synthesized siRNAs. To compare the gene silencing effects of siRNA cocktails generated by RNase III versus individual chemically synthesized siRNAs, we transfected HeLa cells with siRNAs targeting GAPDH generated by both methods at 50 nM, 25 nM and 12.5 nM final concentration. The resulting protein levels were examined 48 hours after transfection. siRNAs prepared by both methods efficiently reduced GAPDH protein levels in a dose dependent manner, although higher concentrations of RNase III-generated siRNAs were required to maximally reduce GAPDH expression levels (data not shown). This finding is not surprising given the fact that siRNA cocktails generated by RNase III consist of both effective and ineffective siRNAs.


Conclusion
RNase III digestion of dsRNA is a robust, fast, and cost effective method
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