Please use this identifier to cite or link to this item: https://hdl.handle.net/1822/22233

TitleOptimizing a qPCR gene expression quantification assay for S. epidermidis biofilms : a comparison between commercial kits and a customized protocol
Author(s)França, Ângela
Freitas, Ana Isabel Costa
Henriques, Ana Filipa Frutuoso Mendes
Cerca, Nuno
Issue date2012
PublisherPublic Library of Science
JournalPLoS ONE
Abstract(s)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.
TypeArticle
URIhttps://hdl.handle.net/1822/22233
DOI10.1371/journal.pone.0037480
ISSN1932-6203
Publisher versionwww.plosone.org
Peer-Reviewedyes
AccessOpen access
Appears in Collections:CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series

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