Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing.
RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.
Myers
Jason W
JW
Department of Molecular Pharmacology, 269 Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5174, USA. jmyers@stanford.edu
Jones
Joshua T
JT
Meyer
Tobias
T
Ferrell
James E
JE
D002460:Cell Line; D004722:Endoribonucleases; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D043244:Ribonuclease III; D014162:Transfection / Q000379:methods
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D008156:Luciferases; D004722:Endoribonucleases; D043244:Ribonuclease III