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Dicer vs. RNase III for Preparation of siRNA Cocktails

t enzyme, requiring long digestion times (12-16 hour). In contrast, RNase III digestions take only an hour. For these reasons, Ambion has chosen RNase III for its Silencer siRNA Cocktail Kit (RNase III), which includes reagents for preparing siRNA cocktails to 20 different genes.

Figure 1. 12-15 bp RNase III Digestion Products Elicit Silencing. A 200 bp GAPDH dsRNA (30 g) was digested with RNase III (30 U) for 1 hour at RT. Digestion products were run on a 15% non-denaturing acrylamide gel and the 12-15 bp products were excised, eluted, and ethanol precipitated. A sample was run on a 15% non-denaturing acrylamide gel for visualization (A). HeLa cells were transfected with 100 nM of the 12-15 bp RNase III generated GAPDH siRNAs or a 21 bp chemically synthesized GAPDH siRNA. GAPDH protein levels were monitored by immunofluorescence 48 hours after transfection (B) and the resulting images were quantitated (C).

Figure 2. Potency and Toxicity of siRNA Cocktails.(A) HeLa cells were transfected 24 h after plating with 50 nM, 25 nM, or 12.5 nM final concentration of either RNase III generated, Dicer generated, or chemically synthesized siRNAs to GAPDH. The cells were harvested after 48 h and the reduction in protein levels was examined by immunofluorescence microscopy. Fluorescent signal was quantitated and normalized for cell number. The level of GAPDH in the cells was reduced in a dose dependent manner for all methods of siRNA production, with a 94%, 92%, or 91% reduction being achieved at 50 nM final siRNA concentration, respectively. (B) Duplicate samples from Panel A were examined for cellular toxicity using a Tr
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