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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 inefficien
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