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Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression

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The Genetic Manipulation of Staphylococci

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1373))

Abstract

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a “two-step” qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression.

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Acknowledgement

This work was supported in part by a sub-award to K.C.R. from NIAID 1P01AI083211.

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Authors and Affiliations

  1. Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, FL, 32611-0700, USA

    April M. Lewis & Kelly C. Rice

Authors
  1. April M. Lewis
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  2. Kelly C. Rice
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Corresponding author

Correspondence to Kelly C. Rice .

Editor information

Editors and Affiliations

  1. University of Kansas Medical Center, Kansas City, Kansas, USA

    Jeffrey L. Bose

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Lewis, A.M., Rice, K.C. (2014). Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression. In: Bose, J. (eds) The Genetic Manipulation of Staphylococci. Methods in Molecular Biology, vol 1373. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_193

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  • DOI: https://doi.org/10.1007/7651_2014_193

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3157-6

  • Online ISBN: 978-1-4939-3158-3

  • eBook Packages: Springer Protocols

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