Lc480 real time cycler

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.

Pneumococci were grown as for the ICP–MS analyses, then 500 μl of the culture was mixed with 1 ml of RNA Protect (Qiagen). RNA was extracted and purified using an RNeasy Protect Bacteria Mini Kit (Qiagen) after enzymatic lysis using lysozyme and mutanolysin, all according to the manufacturer’s instructions. DNase I treatment (Roche) was performed followed by quantitative reverse transcription PCR using SuperScript III (Invitrogen) with a Roche LC480 Real-Time Cycler. The transcription levels of genes analysed were normalized to those obtained for 16S rRNA. Primer sequences are in Supplementary Table 6.

Strains were initially grown overnight on BA plates at 37°C with 5% CO₂. Cells were harvested, washed, and resuspended in 1 mL of CDM + 0.5% glucose or raffinose to a final OD₆₀₀ of 0.2. Suspensions were incubated at 37°C with 5% CO₂ for 30 min. RNA was extracted using a Qiagen RNeasy Minikit as per the manufacturer’s instructions. Differences in levels of gene expression were determined using one-step relative real-time qRT-PCR in a Roche LC480 real-time cycler, as previously described (Minhas et al., 2019 (link)). The specific primers used for the different genes are listed in Table 2 and were used at a final concentration of 200 nM per reaction. Primers specific for gyrA mRNA were used an internal control. Amplification data were analyzed using the comparative critical threshold (2^-∆∆CT) method (Livak and Schmittgen, 2001 (link)). Assays were performed in triplicate with a minimum of two independent experiments. Statistical analyses were performed using two-tailed Student’s t test; P values < 0.05 were defined as statistically significant.