RNA interference (RNAi) with RNase III-prepared siRNAs

Methods Mol Biol. 2004;252:471-82. doi: 10.1385/1-59259-746-7:471.

Abstract

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have varying silencing capacities, several different siRNAS typically must be screened to obtain a region that will effectively silence the gene of interest. However, RNA interference with synthetic siRNA is inefficient and cost-intensive, especially for large, functional genomic studies. Here, we describe the use of E. coli endoribonuclease III to cleave double-stranded RNA (dsRNA) into esiRNA (endoribonuclease-prepared siRNA) that can target multiple sites within an mRNA. EsiRNA mediates effective RNA interference with no apparent nonspecific effects in cultured mammalian cells. Since the whole gene can be used at once, screening for an active siRNA for an individual gene is eliminated. Because of its simplicity and potency, this approach is useful for large-scale analysis of mammalian gene function.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA Primers
  • Escherichia coli / enzymology
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • RNA Interference*
  • RNA, Double-Stranded / chemical synthesis
  • RNA, Double-Stranded / metabolism
  • RNA, Small Interfering / biosynthesis*
  • Recombinant Fusion Proteins / metabolism
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*

Substances

  • DNA Primers
  • RNA, Double-Stranded
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Glutathione Transferase
  • Ribonuclease III