One step rna reagent

Overnight bacterial cultures were diluted 1:100 in fresh LB broth and grown at 37°C to midlogarithmic phase (A₆₀₀ = 0.5–0.6). Total RNA was extracted from S. Typhi isolates using One Step-RNA Reagent (Bio-Basic: BS410A) and treated with DNase I (Thermo Fischer, USA). The RNA samples were quantified and 0.5 μg of DNase-treated RNA was used for cDNA synthesis using the mixture of single-stranded random 6-nucleotide primers and reverse transcriptase in cDNA Synthesis Kit (Thermo Fisher, USA). qRT-PCR was then performed to measure the expression of the styMdtM gene in S. Typhi isolates relative to that of the enolase housekeeping gene. A total volume of 25 μL reaction mixture was prepared by mixing 2.5 μL cDNA (~25 ng) with 12.5 μL SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA), 0.25 μL each of the forward and reverse primers (Table S1; 0.25 pmol), and 9.5 μL deionized water. The used PCR conditions were: 4 minutes at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 45 seconds at 72°C. qRT-PCR was performed in 96-well microtiter plates using an iQ5 thermal cycler (Bio-Rad, USA). Experiments were carried out in triplicate. At the end of every run, a melting curve analysis was performed. Results were analyzed with the ΔΔCt method^{17 (link)}, and the enolase gene (STY3081) was used to normalize the corresponding cycle threshold (Ct) values.