Quantitative Transcriptional Analysis of L. monocytogenes

Total L. monocytogenes RNA was extracted from mid-exponential cultures (OD₆₀₀≈ 0.2–0.3 for BHI media) or intracellular bacteria (t = 4 h) using RNease mini kit (Qiagen). RNA samples were reverse transcribed using ImProm-II^TM reverse transcription system (Promega) and specific cDNAs quantified by real-time PCR (RT-QPCR) as previously described [5 (link)] using Step One Plus real-time PCR apparatus and Step One V2.3 software (Applied Biosystems). The PCR signal was monitored using TaqMan probes for the PrfA-regulated genes hpt and actA, and Power SYBR Green master mix (Applied Biosystems) and gene-specific primers for fosX. Transcription values of the target genes were normalized using the housekeeping genes rpoB and ldh. Fold-changes in fosX expression were determined by the 2^–ΔΔCT method. The oligonucleotides used are shown in S4 Table.