In the current study, various steps in the real-time reverse transcription PCR (real-time RT-PCR) method for determination of RNA expression levels starting from different numbers of Listeria monocytogenes cells were evaluated and optimized. Our results showed that the RNA isolation method as well as the cDNA synthesis may influence the sensitivity of the procedure. For high bacterial cell numbers (10(9) bacterial cells), the RNAqueous kit and the RNeasy Mini kit were equally useful, whereas for low bacterial cell numbers (<or=10(7) bacterial cells) the RNAqueous-Micro kit was found to be the most sensitive RNA isolation kit. For cDNA synthesis, the use of random hexamers and an incubation time of 90 min with the Multiscribe RT-enzyme resulted in the highest efficiency of conversion of RNA into cDNA. To compare RNA levels of different L. monocytogenes strains, it is necessary to analyse the expression levels at the same point in the growth phase and to have a 100% matching of the primers for all tested strains to obtain reliable results. In general, our results showed that real-time RT-PCR needs to be optimized to obtain reliable and accurate data and that many factors can influence the outcome of the real-time RT-PCR data.