( Both recombinant human ...)Dicer vs. RNase III for Preparation of siRNA Cocktails

Both recombinant human Dicer and Escherichia coli RNase III can be used in vitro to cleave long dsRNA, producing siRNA cocktails that are effective mediators of gene silencing (1-6). Dicer is an endoribonuclease that contains RNase III domains and is the enzyme responsible for cleavage of long dsRNAs to siRNA in the endogenous RNAi pathway. The siRNAs produced by Dicer are 21-23 bp in length and contain 3' di-nucleotide overhangs with 5'-phosphate and 3'-hydroxyl termini (7-11). E. coli RNase III is involved in the maturation and degradation of diverse cellular, phage, and plasmid RNAs (12-14). Also responsible for digesting long dsRNA, its cleavage products range from ~12 to 15 bp in length with termini identical to those produced by Dicer (3).

Ambion scientists have spent considerable time comparing the silencing effects of Dicer and RNase III generated siRNA cocktails. Although RNase III does produce shorter cleavage products than Dicer, these shorter products are active mediators of gene silencing when transfected into cells (Figure 1; see also Inducing RNAi with siRNA Cocktails Generated by RNase III). Indeed, no difference in efficacy has been found between RNase III and Dicer generated cocktails (Figure 2). Cytotoxic and nonspecific effects are low for both methods (Figures 2B and 3). Because Dicer is more difficult to overexpress and purify than RNase III, its price is usually much higher. In addition, purified Dicer is a rather inefficient 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 Trypan Blue Exclusion Assay. Background levels of cell death of 2% were recorded in nontransfected cells; none of the transfected samples showed significantly higher levels of cellular toxicity.

Figure 3. RNase III Generated siRNA Cocktails Show Specificity for Silencing. HeLa cells were transfected with 100 nM RNase III generated siRNAs to GAPDH. Immunofluorescence analysis of GAPDH, La, c-MYC, Cdk-2, Ku-90, and -actin was performed 48 hours post transfection and subsequently quantitated.

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Cat# Product Name Size 1625 Silencer siRNA Cocktail Kit (RNase III) 20 rxns
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