Overnight cultures of WT (in the absence or presence of 50 µM epinephrine or norepinephrine) and ΔqseC, ΔqseE and ΔqseEC strains were diluted 1:100 and grown aerobically at 37°C in low-glucose Dulbecco’s modified Eagle medium (DMEM) (Gibco) to the exponential-growth phase (optical density at 600 nm [OD600] = 0.7). RNA was extracted from three biological samples using a RiboPure bacterial RNA isolation kit (Ambion) following the manufacturer’s guidelines. The primers used in the real-time assays were designed using Primer Express v1.5 (Applied Biosystems) (63 (link)) and were validated for amplification efficiency and template specificity. Quantitative real-time PCR (qRT-PCR) was performed as previously described (21 (link)) in a one-step reaction using an ABI 7500 sequence detection system (Applied Biosystems). Data were collected using ABI Sequence detection 1.2 software (Applied Biosystems).
All data were normalized to an rpoA (RNA polymerase subunit A) endogenous control and analyzed using the comparative cycle threshold (CT) method. Virulence gene expression was presented as fold changes over the WT expression level. Error bars indicate the standard deviations of the fold change values. The Student unpaired t test was used to determine statistical significance.
All data were normalized to an rpoA (RNA polymerase subunit A) endogenous control and analyzed using the comparative cycle threshold (CT) method. Virulence gene expression was presented as fold changes over the WT expression level. Error bars indicate the standard deviations of the fold change values. The Student unpaired t test was used to determine statistical significance.
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