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A rapid and sensitive assay for quantification of siRNA efficiency and specificity

Biological Procedures Online volume 7, pages 1–7 (2005)Cite this article

Abstract

RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin β4 (Tβ4); determined their effectiveness at silencing Tβ4 and have both excluded off-target silencing of the Tβ4 homologue Thymosin β10 (Tβ10) and demonstrated partial knockdown of Tβ10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.

References

  1. Jackson A, Bartz S, Scheller J, Kobayashi S, Burchard J, Mao M, Li B, Cavet G, Linsley P. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 2003; 21:635–637.

    Article  PubMed  CAS  Google Scholar 

  2. Bridge A, Pebernard S, Ducraux A, Nicoulaz A-L, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nature Genetics 2003;34:263–264.

    Article  PubMed  CAS  Google Scholar 

  3. Martinez LA, Naguibneva I, Lehrmann H, Vervisch A, Tchenio T, Lozano G, Harel-Bellan A. Synthetic small inhibiting RNAs: Efficient tools to inactivate oncogenic mutations and restore p53 pathways. PNAS 2002; 99:14849–14854.

    Article  PubMed  CAS  Google Scholar 

  4. Semizarov D, Frost L, Sarthy A, Kroeger P, Halbert DN, Fesik SW. Specificity of short interfering RNA determined through gene expression signatures. PNAS 2003; 100:6347–6352.

    Article  PubMed  CAS  Google Scholar 

  5. Chi JT, Chang HY, Wang NN, Chang DS, Dunphy N, Brown PO. Genomewide view of gene silencing by small interfering RNAs. PNAS 2003; 100:6343–6346.

    Article  PubMed  CAS  Google Scholar 

  6. Persengiev SP, Zhu XIAO, Green MR. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 2004; 10:12–18.

    Article  PubMed  CAS  Google Scholar 

  7. Snove J, Holen T. Many commonly used siRNAs risk off-target activity. Biochemical and Biophysical Research Communications 2004; 319:256–263.

    Article  PubMed  CAS  Google Scholar 

  8. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. PNAS 2004;101:1892–1897.

    Article  PubMed  CAS  Google Scholar 

  9. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of Mammalian MicroRNA Targets. Cell 2003; 115:787–798.

    Article  PubMed  CAS  Google Scholar 

  10. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS. MicroRNA targets in Drosophila. Genome 2003; Biol 5:R1.

  11. Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cellderived embryos recapitulates a genetic null phenotype. Nature Biotechnology 2003; 21:559–561.

    Article  PubMed  CAS  Google Scholar 

  12. Huff T, Muller C, Otto A, Netzker R, Hannappel E. β-thymosins, small acidic peptides with multiple functions. Int J Biochem & Cell Biol 2001; 33:205–220.

    Article  CAS  Google Scholar 

  13. Hannon G. RNA Interference. Nature 2002; 418:244–251.

    Article  PubMed  CAS  Google Scholar 

  14. Cui W, Ning J, Naik UP, Duncan MK. OptiRNAi, an RNAi design tool. Computer Methods and Programs in Biomedicine 2004; 75:67–73.

    Article  PubMed  Google Scholar 

  15. Myslinski E, Ame JC, Krol A, Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucl Acids Res 2001; 29:2502–2509.

    Article  PubMed  CAS  Google Scholar 

  16. Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for mutations and chemical modifications in a siRNA. Nucl Acids Res 2003; 31:589–595.

    Article  PubMed  CAS  Google Scholar 

  17. Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411:494–498.

    Article  PubMed  CAS  Google Scholar 

  18. Kumar R, Conklin DS, Mittal V. High-Throughput Selection of Effective RNAi Probes for Gene Silencing. Genome Res 2003; 13:2333–2340.

    Article  PubMed  CAS  Google Scholar 

  19. Zeng Y, Yi R, Cullen BR. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. PNAS 2003; 100:9779–9784.

    Article  PubMed  CAS  Google Scholar 

  20. Holen T, Amarzguioui M, Wiiger MT, Babaie E, Prydz H. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucl Acids Res 2002; 30:1757–1766.

    Article  PubMed  CAS  Google Scholar 

  21. Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature 2004;431:371–378.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Molecular Medicine Unit, Institute of Child Health, 30 Guilford Street, WC1N 1EH, London, United Kingdom

    Nicola Smart, Peter James Scambler & Paul Richard Riley

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  1. Nicola Smart
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  2. Peter James Scambler
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  3. Paul Richard Riley
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Correspondence to Paul Richard Riley.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Smart, N., Scambler, P.J. & Riley, P.R. A rapid and sensitive assay for quantification of siRNA efficiency and specificity. Biol. Proced. Online 7, 1–7 (2005). https://doi.org/10.1251/bpo99

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  • DOI: https://doi.org/10.1251/bpo99

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Biological Procedures Online

ISSN: 1480-9222

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