Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

Nucleic Acids Res. 2004 Feb 9;32(3):936-48. doi: 10.1093/nar/gkh247. Print 2004.


In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Chick Embryo
  • Cricetinae
  • Drosophila / cytology
  • GC Rich Sequence
  • Genetic Vectors
  • Humans
  • Luciferases / genetics
  • Mice
  • Models, Genetic
  • RNA Interference*
  • RNA, Small Interfering / chemistry*
  • RNA, Small Interfering / genetics
  • Transfection


  • RNA, Small Interfering
  • Luciferases