Abstract
Transcriptomic studies of microorganisms are dependent upon the efficiency of the RNA extraction procedure. In this study, we compared different methods used to disrupt bacterial cells that are frequently described in the literature, such as mechanical (sonication, bead beating) and enzymatic (lysozyme or lysostaphin digestion) disruption. Factorial designs and ANOVA procedureswere used to compare statistically the efficiency of these protocols on Staphylococcus aureus. The results were assessed in terms of quality and quantity of RNA extract suitable for further quantitative reverse transcriptase PCR (qRT-PCR) analysis. We selected a simple, rapid (in less than four hours) and sensitive RNA extraction/purification protocol based on lysostaphin treatment, followed by a bead-beating procedure. This method allowed an excellent recovery (> 85%) of 16S rRNA from over a wide range of CFU (109 to 102 CFU·mL−1), efficient on different S. aureus strains in both exponential and stationary growth phases in pure culture. Application of the protocol for the examination of artificially contaminated Camembert cheese achieved sensitivities of 1.1 × 102 copies of the 16S rRNA gene·g−1 of cheese. This protocol constitutes an essential tool for gene expression studies of S. aureus in Camembert cheese.
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