Rational design and in vitro and in vivo delivery of Dicer substrate siRNA

The Biotechnology Centre of Oslo, Gaustadalleen 21, 0349 Oslo, Norway.
Nature Protocols (Impact Factor: 7.78). 06/2006; 1(2):508-517. DOI: 10.1038/nprot.2006.72
Source: PubMed

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.

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