C1000 touch cfx384 machine

L. monocytogenes was grown in BHI at 37°C under constant agitation until reaching an optical density at 600 nm (OD₆₀₀) of 0.9, pelleted, and frozen at −80°C. RNAs were extracted as previously described (72 (link)). For each sample, 10 µg of RNA was treated with DNase I (Turbo DNA-free kit, Ambion). RNAs (500 ng) were reverse transcribed with Quantiscript reverse transcriptase (QuantiTect reverse transcription [RT] kit; Qiagen). Quantitative RT-PCRs (qRT-PCRs) were carried out using oligonucleotides qPCR-orfX-for, qPCR-orfX-rev, qPCR-gyrA-for, qPCR-gyrA-rev, qPCR-rpoB-for, and qPCR-rpoB-rev (Table 1), and the products quantified with SYBR green master mix on a C1000 Touch CFX384 machine (Bio-Rad). The expression levels of orfX were normalized to those of L. monocytogenes rpoB and gyrA genes, and the fold changes in expression were calculated using the cycle threshold (ΔΔCT) method. All samples were evaluated in triplicate and in at least three independent experiments.