Rational design and in vitro and in vivo delivery of Dicer substrate siRNA.
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.
Amarzguioui
Mohammed
M
The Biotechnology Centre of Oslo, Gaustadalleen 21, 0349 Oslo, Norway. moamarzg@biotek.uio.no
Lundberg
Patric
P
Cantin
Edouard
E
Hagstrom
James
J
Behlke
Mark A
MA
Rossi
John J
JJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection
D034741:RNA, Small Interfering; D043244:Ribonuclease III