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Silencing Gene Expression with Dicer-Generated siRNA Pools

  • Protocol
Book cover RNA Silencing

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 309))

Abstract

With the explosion of genomic information, there is an increasing need to analyze gene function in a high-throughput fashion. This makes reverse genetic approaches extremely attractive; however, in most mammalian and vertebrate systems it has been difficult and time-consuming to develop a cellular model deficient in one or more proteins. The discovery of RNA interference (RNAi) has made loss-of-function studies relatively quick and easy and amenable to high-throughput formats (1–3). Double-stranded RNAs (dsRNAs) 21 nucleotides in length, known as small interfering RNAs (siRNAs), are introduced into the cytosol, where they are unwound (4), allowing the antisense strand to interact in a sequence-specific manner with the complementary mRNA (5). Binding of the antisense strand to the target mRNA triggers cleavage of the mRNA (6). siRNA-mediated gene silencing is very specific, most likely because of the high specificity of nucleotide base-pairing.

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Acknowledgments

We thank Josh Jones, Delquin Gong, David Hendrickson, Ari Firestone, and members of the Ferrell lab for insightful discussion about and validation of the techniques presented here. Also, we thank Knut Madden, Adam Harris, Michaeline Bunting, Kerry Lowrie, Joe O’Connor, and Byung-in Lee for help with this protocol. The NIH grant GM46383 supports our work in this area.

Author information

Authors and Affiliations

  1. Department of Molecular Pharmacology, Stanford University Medical School, Stanford, CA

    Jason W. Myers

  2. Departments of Molecular Pharmacology and Biochemistry, Stanford University Medical School, Stanford, CA

    James E. Ferrell Jr.

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  1. Jason W. Myers
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  2. James E. Ferrell Jr.
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Editor information

Editors and Affiliations

  1. Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT

    Gordon G. Carmichael

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© 2005 Humana Press Inc.

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Myers, J.W., Ferrell, J.E. (2005). Silencing Gene Expression with Dicer-Generated siRNA Pools. In: Carmichael, G.G. (eds) RNA Silencing. Methods in Molecular Biology™, vol 309. Humana Press. https://doi.org/10.1385/1-59259-935-4:093

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  • DOI: https://doi.org/10.1385/1-59259-935-4:093

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-436-4

  • Online ISBN: 978-1-59259-935-6

  • eBook Packages: Springer Protocols

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