Design, Manufacture, and Assay of the Efficacy of siRNAs for Gene Silencing

  • Louise A. Dawson
  • Badar A. Usmani
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)


Small interfering RNAs (siRNAs) have been widely exploited for nucleotide-sequence-specific posttranscriptional gene silencing, as a tool to investigate gene function in eukaryotes, and they hold promise as potential therapeutic agents. Conventionally designed siRNAs are 21-mers with symmetric 2-nt 3′ overhangs that mimic intermediates (microRNAs or miRNAs) of the natural processing of longer dsRNA (double-stranded RNA). siRNAs are sequences with full c omplementarity to their target mRNA and can be generated by either chemical synthesis or processing of shRNAs (short hairpin RNAs) transcribed from DNA vectors. To minimize off-target effects, any homology to nontarget mRNA can be verified using the expressed sequence tag (EST) database for the relevant organism. Here, we provide a practical guide and an overview to the design and selection of effective and specifc siRNAs.


small interfering RNA gene silencing endothelin-converting enzyme prostate cancer invasion stromal-epithelial interactions 



This work was supported by Yorkshire Cancer Research (YCR) and Prostate Cancer Research Foundation (PCRF).


  1. 1.
    1. Fire A et al (1998) Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefPubMedGoogle Scholar
  2. 2.
    2. Guo S, Kemphues KJ (1995) par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611–620CrossRefPubMedGoogle Scholar
  3. 3.
    3. Jorgensen R (1990) Altered gene expression in plants due to trans activations between homologous genes. Trends Biotechnol 8:340–344CrossRefPubMedGoogle Scholar
  4. 4.
    4. Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937–946CrossRefPubMedGoogle Scholar
  5. 5.
    5. Elbashir SM et al (2001) Duplexes of 21- nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498CrossRefPubMedGoogle Scholar
  6. 6.
    6. Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191–3197CrossRefPubMedGoogle Scholar
  7. 7.
    7. Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296CrossRefPubMedGoogle Scholar
  8. 8.
    8. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366CrossRefPubMedGoogle Scholar
  9. 9.
    9. Nykanen A, Haley B, Zamore PD (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309–321CrossRefPubMedGoogle Scholar
  10. 10.
    10. Paddison PJ, Caudy AA, Sachinandam R, Hannon GJ (2004) Short hairpin activated gene silencing in mammalian cells. Methods Mol Biol 265:85–100PubMedGoogle Scholar
  11. 11.
    11. Song E et al (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nature Med 9:347–51CrossRefPubMedGoogle Scholar
  12. 12.
    12. Ui-Tei K, Naito Y, Saigo K (2006) Essential notes regarding the design of functional siRNAs for efficient mammalian RNAi. J Biomed Biotechnol 65052: 1–8CrossRefGoogle Scholar
  13. 13.
    13. Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199–213CrossRefPubMedGoogle Scholar
  14. 14.
    14. Kiermer V (ed). (2006) Focus on RNA interference—a user's guide. Nature Methods 3:669–719CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Louise A. Dawson
    • 1
  • Badar A. Usmani
    • 1
  1. 1.Institute of Cellular and Molecular BiologyUniversity of LeedsLeedsUK

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