Protocol abstract
Nature Protocols 1, - 508 - 517 (2006)
Published online: 27 June 2006 | doi:10.1038/nprot.2006.72
Subject Categories: Computational and theoretical biology | Genetic analysis | Genomics and proteomics | Isolation, purification and separation | Model organisms | Nucleic acid based molecular biology
Rational design and in vitro and in vivo delivery of Dicer substrate siRNA
Mohammed Amarzguioui1, Patric Lundberg2, Edouard Cantin2, James Hagstrom3, Mark A Behlke4 & John J Rossi5
Abstract
RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21–25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.
- The Biotechnology Centre of Oslo, Gaustadalleen 21, 0349 Oslo, Norway.
- Division of Virology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010, USA.
- Mirus Bio Corporation, 505 S. Rosa Road, Suite 104, Madison, Wisconsin 53719-1267, USA.
- Integrated DNA Technologies, 1710 Commercial Park, Coralville, Iowa 52241, USA.
- Division of Molecular Biology and Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010, USA.
Correspondence to: Mohammed Amarzguioui1 e-mail: moamarzg@biotek.uio.no
Correspondence to: John J Rossi5 e-mail: jrossi@bricoh.edu
nature-products
A-Z product listing
- DMEM(Cellgro)
- HEPES(Irvine Scientific)
- L-glutamine(Cellgro)
- Lipofectamine 2000(Invitrogen)
- Low-endotoxin FBS(Omega Scientific)
- OPTI-MEM I(Invitrogen Gibco)
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