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Gene Expression Analysis in Bacteria by RT-qPCR

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Quantitative Real-Time PCR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2065))

Abstract

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.

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Acknowledgments

Work in our group is supported by grants from Fundação de Amparo à Pesquisa no Estado da Bahia (FAPESB), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Authors and Affiliations

  1. Post-Graduate Program in Biotechnology, Institute of Health Sciences, Federal University of Bahia, Salvador, BA, Brazil

    Danilo J. P. G. Rocha, Thiago L. P. Castro, Eric R. G. R. Aguiar & Luis G. C. Pacheco

Authors
  1. Danilo J. P. G. Rocha
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  2. Thiago L. P. Castro
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  3. Eric R. G. R. Aguiar
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  4. Luis G. C. Pacheco
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Corresponding author

Correspondence to Luis G. C. Pacheco .

Editor information

Editors and Affiliations

  1. Molecular Diagnostics, IRCCS, Istituto Giannina Gaslini, Genova, Italy

    Roberto Biassoni

  2. ASL3 Sistema Sanitario Regione Liguria, S.C. Laboratorio d’Analisi, Genova, Italy

    Alessandro Raso

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Rocha, D.J.P.G., Castro, T.L.P., Aguiar, E.R.G.R., Pacheco, L.G.C. (2020). Gene Expression Analysis in Bacteria by RT-qPCR. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 2065. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9833-3_10

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  • DOI: https://doi.org/10.1007/978-1-4939-9833-3_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9832-6

  • Online ISBN: 978-1-4939-9833-3

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