Quantification of CXCL8 Expression in Caco-2 Cells

Caco-2 cells were incubated with either EHEC at different multiplicity of infection (MOI range of 1–100) or S. aureus for 2 h, 4 h and 8 h. Total RNA was extracted using the RNeasy Mini Kit following the manufacturer’s instructions [42 (link)]. RNA concentration and purity were determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 25 ng of total RNA were reverse transcribed using the SuperScript^® III First-Strand Synthesis SuperMix and amplified using the EXPRESS SYBR^® GreenER™ qPCR SuperMix according to the manufacturer’s protocol at a final volume of 20 µL. Real-time PCR reactions were conducted on a RotorGene Q Qiagen Real-Time PCR System (QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden), with an initial 5 min incubation at 60 °C, then 2 min at 95 °C, followed by 40 cycles of amplification: 95 °C for 15 s and 60 °C for 1 min, and examined by Rotor-Gene Real-Time Analysis Software 6.0 (QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden, Germany). Primer concentration was 500 nM. The following primers were used: CXCL8 forward 5′- CCACCGGAAGGAACCATCTC-3′ reverse 5′- GGCAAAACTGCACCTTCACA-3′; RPL13A forward 5′- CACCCTGGAGGAGAAGAGGA-3′, reverse 5′- CCGTAGCCTCATGAGCTGTT-3′. Changes in gene expression were calculated by the 2^−ΔΔCt formula using RPL13A as reference gene.