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J Microbiol Methods. 2004 Dec;59(3):363-70.

Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth.

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Laboratoire de Recherches sur les Biomatériaux et Biotechnologies, Université du Littoral-Côte d'Opale, Inserm-ERI002, LR2B, BP 120, F-62327 Boulogne-sur-mer cedex, France.


By real-time quantitative PCR (RTQ-PCR), two different standardisation methods were used to quantify expression of three target genes (RNAII and RNAIII transcripts of agr locus and ica transcript of icaADBC locus): (i) a relative quantification, using a transcript of three housekeeping genes (gyrase A, gyrA; guanylate kinase, gmk and 16S rRNA, 16S) as internal standard, and (ii) an absolute quantification, using cloned sequences of the target genes in known concentrations as external standards. To determine the efficiency and reliability of these two methods, the gene expressions were studied during the growth of a clinical isolate of Staphylococcus aureus. Between 3 and 20 h after inoculation, target gene transcription was analysed using LightCycler Apparatus, LC Data Analysis software and RelQuant software for relative quantification (Roche). For all target genes, the expression profiles obtained with gyrA or gmk as internal standards remained almost identical. However, these profiles varied between each other depending on the standard gene. Due to their important expression variations during growth phases, these two housekeeping genes seem inappropriate to be used as internal standards. The absolute quantification of the three transcripts of interest gave results similar to their relative quantification expressed versus 16S rRNA. Therefore, our study suggests the suitable use of 16S rRNA as internal standard in RTQ-PCR quantification of staphylococcal gene expression during the stationary phase of growth.

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