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

Nat Protoc. 2006;1(2):508-17. doi: 10.1038/nprot.2006.72.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Macrophages / metabolism*
  • Mice
  • RNA Interference*
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism*
  • Ribonuclease III / metabolism*
  • Substrate Specificity
  • Time Factors
  • Transfection

Substances

  • RNA, Small Interfering
  • Ribonuclease III