Determination of interferon-β mRNA expression by quantitative RT-PCR

Cell mRNA was extracted 6 h post-infection (p.i) according to the manufacturer’s instructions (Qiagen) and cDNA generated using the Quantitect cDNA Synthesis Kit (Qiagen) as previously described [32]. Incubation time was chosen based on maximal expression of interferon (IFN)-β by DCs following S. suis infection [32]. Real-time qPCR was performed on the CFX-96 Touch Rapid Thermal Cycler System (Bio-Rad) using 250 nM of primers (Integrated DNA technologies) and the SsoFast Evagreen Supermix Kit (Bio-Rad). Cycling conditions were 3 min of polymerase activation at 98°C, followed by 40 cycles at 98°C for 2 sec and 57°C for 5 sec. Melting curves were generated after each run to confirm presence of a single PCR product. The primer sequences used in this study are shown in Table 2 and were verified to have reaction efficiencies between 90% and 110%. The reference genes Atp5b and Gapdh, determined to be the most stably expressed using the algorithm geNorm, were used to normalize data. Fold changes in gene expression were calculated using the quantification cycle threshold (Cq) method using the CFX software manager v.3.0 (Bio-Rad). Samples from mock-infected cells served as calibrators. IFN-β was measured by RT-qPCR in order to compare with published results [32].