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Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis

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Abstract

The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.

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Acknowledgments

DJPR is recipient of a scholarship from the National Council for Scientific and Technological Development of Brazil (CNPq). CSS is recipient of a scholarship from the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB). Work at our group is supported by grants from the Brazilian Research Funding Agencies FAPESB, CAPES and CNPq.

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Authors and Affiliations

  1. Institute of Health Sciences, Federal University of Bahia, Salvador, BA, Brazil

    Danilo J. P. Rocha, Carolina S. Santos & Luis G. C. Pacheco

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  1. Danilo J. P. Rocha
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  2. Carolina S. Santos
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  3. Luis G. C. Pacheco
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Correspondence to Luis G. C. Pacheco.

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Rocha, D.J.P., Santos, C.S. & Pacheco, L.G.C. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis. Antonie van Leeuwenhoek 108, 685–693 (2015). https://doi.org/10.1007/s10482-015-0524-1

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