title: Single-pot enzymatic synthesis of Dicer-substrate siRNAs.
abstract: We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25-27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Keelan Z
authors.initials: KZ
authors.lastname: Guiley
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Pratt
authors.forename: Ian J
authors.initials: IJ
authors.lastname: MacRae
mesh_terms: D000818:Animals; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D020868:Gene Silencing; D017930:Genes, Reporter; D016829:Giardia lamblia / Q000201:enzymology; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D034741:RNA, Small Interfering; D008156:Luciferases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases