Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR

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Journal Article
Journal of Microbiological Methods, 2007, 70 pp. 355 - 362
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Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of inetrest. The aim of this study was to develop a qyantitative real-time PCR awway to analyse gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Elevn different housekeeping genes were anlysed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk, and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine respectively. Subsequently, these housekeeping egnes were used as internal controls to analyse expression of the multidrug tyransport protei QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qaCR expression was found to be unaffected, reduced or enhanced. This study demonstrated that gene expression including housekeeping genes previously used to normalise qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitanle genes useable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilised as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from s. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transpoerters from s.aureus.
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