qRT-PCR

cDNA was made from 1 μg of RNA using high-capacity RNA-to-DNA master mix (Applied Biosystems). Amplification was performed with SYBR green master mix in a Step One Plus thermal cycler (Applied Biosystems). The primers were designed using Primer Express software. Forty cycles were run with denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 60 °C for 45 s. The gene rpoD was used as a control. See Table ^S2 for primer sequences.

500 ng of RNA was treated with TURBO DNase (Ambion/Life Technologies) according to the manufacturer’s instructions. The RNA was then converted into cDNA using the High Capacity cDNA Conversion Kit (Applied Biosystems/Life Technologies). Thermal cycling was performed using Maxima SYBR Green/ROX qRT-PCR Master Mix (Fermentas) in a Stratagene MX3000p with the following cycling conditions: Preincubation of 95 °C for 10 min followed by 45 cycles of 95 °C for 30 s; 60 °C for 60 s; 72 °C for 60 s. Upon completion, melting curve data was obtained. Data analysis was performed in the MxPro software version 4.1 (Stratagene). RNAIII and hla expression data was normalized to the expression of ileS, which was found to be stably expressed in all strains and conditions. See Table ^S2 for primer sequences.