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Inducing RNAi with siRNA Cocktails Generated by RNase III

Mike W. Byrom, Angie M. Cheng and Lance P. Ford*
Ambion, Inc.
2130 Woodward Street
Austin, TX 78744-1832
*Corresponding author email: lford@ambion.com


Abstract
Small interfering RNA (siRNA) is an extremely effective tool for reducing gene expression in a variety of systems, including mammalian cells. Gene specific siRNAs have typically been made by chemical synthesis. Producing them by chemical synthesis, however, is costly, and while most siRNAs exhibit some effect, only about 25% of those designed reduce target levels by 80% or more (1). A new in vitro procedure that cleaves long dsRNA into a mixture of siRNAs significantly reduces the cost of siRNA preparation. The resulting siRNA population is effective at mediating gene specific silencing and bypasses the need to screen for functional siRNAs.


Introduction
In the RNA interference (RNAi) pathway in C. elegans, Drosophila, and other diverse organisms, long double-stranded RNA (dsRNA) introduced into a cell is cleaved into a mixture of siRNAs by an RNase III-like endoribonuclease (Dicer) producing siRNAs that are 21-23 bp in length and contain 3' dinucleotide overhangs with 5'-phosphate and 3'-hydroxyl termini. These siRNAs mediate degradation of homologous transcripts, leading to gene silencing (2-5; see also "Gene Specifi
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