Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly u...

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Autores principales:	Angela França, Ana I Freitas, Ana F Henriques, Nuno Cerca
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b1d9e7baa5c04a0bbe35b21d05562110
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spelling	oai:doaj.org-article:b1d9e7baa5c04a0bbe35b21d055621102021-11-18T07:17:55ZOptimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.1932-620310.1371/journal.pone.0037480https://doaj.org/article/b1d9e7baa5c04a0bbe35b21d055621102012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22629403/?tool=EBIhttps://doaj.org/toc/1932-6203Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.Angela FrançaAna I FreitasAna F HenriquesNuno CercaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 5, p e37480 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Angela França Ana I Freitas Ana F Henriques Nuno Cerca Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
description	Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.
format	article
author	Angela França Ana I Freitas Ana F Henriques Nuno Cerca
author_facet	Angela França Ana I Freitas Ana F Henriques Nuno Cerca
author_sort	Angela França
title	Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
title_short	Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
title_full	Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
title_fullStr	Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
title_full_unstemmed	Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
title_sort	optimizing a qpcr gene expression quantification assay for s. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/b1d9e7baa5c04a0bbe35b21d05562110
work_keys_str_mv	AT angelafranca optimizingaqpcrgeneexpressionquantificationassayforsepidermidisbiofilmsacomparisonbetweencommercialkitsandacustomizedprotocol AT anaifreitas optimizingaqpcrgeneexpressionquantificationassayforsepidermidisbiofilmsacomparisonbetweencommercialkitsandacustomizedprotocol AT anafhenriques optimizingaqpcrgeneexpressionquantificationassayforsepidermidisbiofilmsacomparisonbetweencommercialkitsandacustomizedprotocol AT nunocerca optimizingaqpcrgeneexpressionquantificationassayforsepidermidisbiofilmsacomparisonbetweencommercialkitsandacustomizedprotocol
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Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.

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