Streptomycin Dose-Dependent Gene Expression

Briefly, the wild-type strain was grown to OD₆₀₀ 0.6–0.8 and then treated with 0.125 and 0.25 μg/ml STR for 2, 4, 6, 12, and 24 h. The broth was collected and the RNA was extracted, as described previously (Yang et al., 2012 (link)). The cDNA was obtained using the HiScript II Q RT kit (Vazyme, Nanjing, China). The qRT-PCR system consisted of 20 μl solution containing 10 μl 2 × SYBR Green qPCR mix, specific primers, and 1 μl of cDNA. The qRT-PCR was performed on Roche 480 instrument as follows: 95°C for 1 min and 40 cycles of 95°C 15 s, 60°C 15 s, and 72°C 30 s. The expression level of each gene was normalized with sigA as an internal reference. Gene relative expression was determined according to the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)).