RNA Extraction and qRT-PCR Analysis of Metal-Stressed Bacteria

For RNA extraction and qRT-PCR analysis, untreated or metal-stressed bacteria (LB supplemented with 400 μM ZnSO₄, 400 μM CuSO₄, or 400 μM ZnSO₄ + 400 μM CuSO₄) were harvested at mid log-phase and lysed in QiaZol (Qiagen) as described previously [55 (link), 56 (link)]. Following the addition of chloroform and phase separation, RNA was extracted and purified using a PureLink RNA Mini Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The total RNA samples were treated with DNase I (Roche) and qRT-PCR performed using the SuperScript III One-Step RT-PCR kit (Thermo Fisher Scientific) on a LC480 Real-Time Cycler (Roche). Transcription levels of genes were corrected to those obtained for GAPDH prior to normalization to the transcription levels observed for untreated A. baumannii cultures. Primer sequences are listed in Additional file 2. Results are the mean (± SEM) of at least three independent experiments, with the statistical significance determined using a one-way ANOVA with Dunnett’s post-test.