Designing functional siRNA with reduced off-target effects

Methods Mol Biol. 2013:942:57-68. doi: 10.1007/978-1-62703-119-6_3.


RNA interference (RNAi) mediated by small interfering RNA (siRNA) is now widely used to knock down gene expression in a sequence-specific manner, making it a powerful tool not only for studying gene functions but also for therapeutic applications. siRNA decreases the expression level of the intended target gene with complete complementarity by cleaving its mRNA. However, the efficacy of each siRNA widely varies depending on its sequence in mammalian cells; only a limited fraction of randomly designed siRNAs is functional. Moreover, off-target silencing effects arise when the siRNA has partial complementarity in the seed region with unintended genes. Here, we describe the rational designing of functional, off-target effect-reduced siRNAs using siDirect 2.0 Web server ( By using the default parameters, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in the RefSeq database.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA Polymerase III / metabolism
  • Genetic Engineering / methods*
  • Humans
  • Internet
  • Mice
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics
  • RNA Interference
  • RNA Stability
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics*
  • Rats
  • Thermodynamics


  • RNA, Small Interfering
  • DNA Polymerase III