Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells

RNA interference (RNAi) has revolutionized functional genomic studies in many model organisms. In particular, the introduction of short double-stranded RNAs (dsRNAs) as mediators of RNAi in mammalian cells has moved loss-of-function studies in these experimental systems to a new plane¹. Now the two most commonly used RNAi triggers in mammalian cells ar chemically or in vitro synthesized short interfeing RNAs (siRNAs)² and in vivio expressed short hairpin RNAs (shRNAs)³. We, and others, have pioneered an alternative technology, which is considerably more cost-efficient and less laborious than siRNAs and shRNA methodologies. Our method is based on the generation of siRNAs by digestion of long dsRNAs with recombinant Escherichia coli RNase III or Dicer^4-8. The resulting endoribonuclease-prepared siRNAs (esiRNAs) have proven to be efficient an specific mediators of RNAi in mammalian cells^9-11. Here we describe a robust and simple protocol for the production of esiRNA (Fig. 1): a cDNA fragment tagged with T7 promoter sequence by PCR is transcribed in vitro to porduce dsRNA. By limited digestion, the long dsRNA is then converted to siRNAs of <30 base pairs (bp) and spin-purified in a single column. Because of its ease, speed and cost-efficiency, this protocol can be easily scaled for the generation of libraries for (sub)genomic screens.