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RNAi-Mediated Knockdown of Protein Expression

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Functional Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1654))

Abstract

RNA interference is an essential method for studying genomic functions of single genes by loss-of-function experiments. Short interfering siRNAs are efficiently transfected into cultured cells to enable RISC-mediated mRNA cleavage and inhibition of translation in a sequence-specific manner. RNAi enables knockdown of single genes and screening for specific cellular processes or outcomes. In this chapter, we describe a detailed universal protocol for lipoplex-mediated siRNA transfection for cell cultures and cell lysis for subsequent RNA or protein analysis. The experimental procedure is described for verification of knockdown and includes cell lysis for mRNA or protein quantification. Important aspects for specific gene silencing and potential pitfalls are discussed.

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Author information

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090, Vienna, Austria

    Volker Baumann, Cornelia Lorenzer, Michael Thell, Anna-Maria Winkler & Johannes Winkler

  2. Department of Cardiology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria

    Johannes Winkler

Authors
  1. Volker Baumann
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  2. Cornelia Lorenzer
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  3. Michael Thell
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  4. Anna-Maria Winkler
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  5. Johannes Winkler
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Corresponding author

Correspondence to Johannes Winkler .

Editor information

Editors and Affiliations

  1. Chair of Biochemistry and Molecular Medicine, Division of Functional Genomics, Faculty of Health, School of Medicine, Center for Biochemical Research and Education ZBAF, Witten/Herdecke University, Witten, Germany

    Michael Kaufmann

  2. Chair of Biochemistry and Molecular Medicine, Division of Functional Genomics, Faculty of Health, School of Medicine, Center for Biochemical Research and Education ZBAF, Witten/Herdecke University, Witten, Germany

    Claudia Klinger

  3. Chair of Biochemistry and Molecular Medicine, Division of Functional Genomics, Faculty of Health, School of Medicine, Center for Biochemical Research and Education ZBAF, Witten/Herdecke University, Witten, Germany

    Andreas Savelsbergh

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Baumann, V., Lorenzer, C., Thell, M., Winkler, AM., Winkler, J. (2017). RNAi-Mediated Knockdown of Protein Expression. In: Kaufmann, M., Klinger, C., Savelsbergh, A. (eds) Functional Genomics. Methods in Molecular Biology, vol 1654. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7231-9_26

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  • DOI: https://doi.org/10.1007/978-1-4939-7231-9_26

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7230-2

  • Online ISBN: 978-1-4939-7231-9

  • eBook Packages: Springer Protocols

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