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Validation of Short Interfering RNA Knockdowns by Quantitative Real-Time PCR

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Protocols for Nucleic Acid Analysis by Nonradioactive Probes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 353))

Abstract

RNA interference (RNAi) is a natural mechanism, that is triggered by the introduction of double-stranded RNA into a cell. The long double-stranded RNA is then processed into short interfering RNA (siRNA) that mediates sequence-specific degradation of homologous transcripts. This phenomenon can be exploited to experimentally trigger RNAi and downregulate gene expression by transfecting mammalian cells with synthetic siRNA. Thus, siRNAs can be designed to specifically silence the expression of genes bearing a particular target sequence. In this chapter, we present methods and procedures for alidating the effects of siRNA-based gene silencing on target gene expression. To illustrate our approach, we use examples from our analysis of a Cancer Gene Library of 278 siRNAs targeting 139 classic oncogenes and tumor suppressor genes (Qiagen Inc., Germantown, MD). Specifically, this library was used for high-throughput RNAi phenotype analysis followed by gene expression analysis to validate gene silencing for siRNA that produced a phenotype. Methods and protocols are presented that illustrate how sequence-specific gene silencing of effective siRNAs are analyzed and validated by quantitative real-time PCR assays to measure the extent of target gene silencing, as well as effects on various gene expression end points.

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Author information

Authors and Affiliations

  1. Molecular Genetics Laboratory, Pharmaceutical Genomics Division, Translational Genomics Institute, Scottsdale, AZ

    Sukru Tuzmen

  2. Knowledge Mining Laboratory, Pharmaceutical Genomics Division, Translational Genomics Research Institute, Scottsdale, AZ

    Jeff Kiefer

  3. Cancer Drug Development Laboratory, Pharmaceutical Genomics Division, Translational Genomics Research Institute, Scottsdale, AZ

    Spyro Mousses

Authors
  1. Sukru Tuzmen
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  2. Jeff Kiefer
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  3. Spyro Mousses
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Editor information

Editors and Affiliations

  1. Gene Technologies, HortResearch Ltd., Mt. Albert, Auckland, New Zealand

    Elena Hilario

  2. Applied Science, Roche Diagnostics, Auckland, New Zealand

    John Mackay

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© 2007 Humana Press Inc.

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Tuzmen, S., Kiefer, J., Mousses, S. (2007). Validation of Short Interfering RNA Knockdowns by Quantitative Real-Time PCR. In: Hilario, E., Mackay, J. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology, vol 353. Humana Press. https://doi.org/10.1385/1-59745-229-7:177

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  • DOI: https://doi.org/10.1385/1-59745-229-7:177

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-430-2

  • Online ISBN: 978-1-59745-229-8

  • eBook Packages: Springer Protocols

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