Abstract
Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched for identification of the most stably expressed genes of a given bacterium. Herein, we searched a RNA-seq dataset from various experiments with the pathogenic bacterium Corynebacterium pseudotuberculosis, grown under different stress conditions, in order to select appropriate candidate reference genes for this species. Nineteen genes involved in maintenance of basic cellular functions, so-called housekeeping genes, were chosen for study and their expression profiles in C. pseudotuberculosis were evaluated throughout all growth conditions. Eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, matched the defined criteria to be included as candidate reference genes. Transcriptional levels of these genes were quantified by RT-qPCR assays after growth of C. pseudotuberculosis under two additional conditions. Expression stability analysis by NormFinder indicated the combination of genes encoding DNA gyrase subunit A (gyrA) and elongation factor P (fusA) as the most suitable for normalization of RT-qPCR studies in C. pseudotuberculosis.
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Acknowledgments
DMC is recipient of a scholarship from 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. We acknowledge technical help from the following laboratories: Labimuno, Lab. Imunofarmacologia, Bioprospector, Lab. Alergia e Acarologia.
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Carvalho, D.M., de Sá, P.H., Castro, T.L.P. et al. Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data. Antonie van Leeuwenhoek 106, 605–614 (2014). https://doi.org/10.1007/s10482-014-0231-3
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DOI: https://doi.org/10.1007/s10482-014-0231-3