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High-Throughput Real-Time PCR for Detection of Gene-Expression Levels

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Cell-Based Assays for High-Throughput Screening

Part of the book series: Methods in Molecular Biology ((MIMB,volume 486))

Summary

While many high-throughput screening campaigns involve the measurement of protein levels or locations, at times it is desirable to measure the levels of gene expression in response to small molecules. Here, we describe a method for capturing mRNA in multiwell plates following compound treatment and measuring gene expression using real-time PCR. This streamlined protocol provides complementary information to conventional phenotypic cell-based assays, and is especially useful in cases where the gene of interest is thought to serve a regulatory function in downstream cellular phenotypes.

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Acknowledgments

We are indebted to the screening staff at the Broad Institute, particularly Stephanie Norton and Jason Burbank, for their help with compound pin-transfer, calibrating and programming robotic equipment, and general advice. We thank Yanhong Ma, Tamara Gilbert, and Daniel Fass for technical advice and expertise. This work has been funded by NIH grant 1R21NS059440 (to Z.A.), and in part with Federal funds from the National Cancer Institute’s Initiative for Chemical Genetics, National Institutes of Health, under Contract No. N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Service, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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Authors and Affiliations

  1. Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Bridget K. Wagner

  2. Cardiovascular Institute at the Beth Israel Deaconess Medical Center Dana-Farber Cancer Institute, Boston, MA, USA

    Zoltan Arany

Authors
  1. Bridget K. Wagner
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  2. Zoltan Arany
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Corresponding author

Correspondence to Bridget K. Wagner .

Editor information

Editors and Affiliations

  1. Broad Institute of Harvard, Chemical Biology Program, Massachusetts Institute of Technology, Cambridge Center 7, Cambridge, 02142, U.S.A.

    Paul A. Clemons

  2. Broad Institute of Harvard, Chemical Biology Program, Massachusetts Institute of Technology, Cambridge Center 7, Cambridge, 02142, U.S.A.

    Nicola J. Tolliday

  3. Broad Institute of Harvard, Chemical Biology Program, Massachusetts Institute of Technology, Cambridge Center 7, Cambridge, 02142, U.S.A.

    Bridget K. Wagner

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Wagner, B.K., Arany, Z. (2009). High-Throughput Real-Time PCR for Detection of Gene-Expression Levels. In: Clemons, P., Tolliday, N., Wagner, B. (eds) Cell-Based Assays for High-Throughput Screening. Methods in Molecular Biology, vol 486. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-545-3_12

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  • DOI: https://doi.org/10.1007/978-1-60327-545-3_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-544-6

  • Online ISBN: 978-1-60327-545-3

  • eBook Packages: Springer Protocols

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