Determination of COX-2 and inducible nitric oxide synthase mRNA levels with quantitative real-time polymerase chain reaction

Total RNA isolation from cells was performed via phenol-guanidine thiocyanate extraction using RNAzol isolation reagent (Sigma-Aldrich), according to the manufacturer’s instructions. Total RNA (1 µg) was reverse-transcribed to cDNA using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche) in a 20 µL reaction mixture. Real-time polymerase chain reaction (PCR) was carried out using a LightCycler Nano System (Roche). To quantify cDNA, qPCR was performed using FastStart Essential DNA probe master mix (Roche). The reaction mixture (15 µL) was prepared in LightCycler 8-tube strips (Roche) and included 10 µL of 2´ Master Reaction Mix (Roche), 4 µL of PCR grade water, 1 µL of catalogue assay kit (kits consist mix of primers and probes for determination of iNOS, COX-2, β-actin), and 5 µL of cDNA. Real-time PCR was performed according to the following conditions: activation of Taq DNA polymerase and DNA denaturation at 95°C for 10 min, followed by 45 amplification cycles for 10 s at 95°C and for 30 s at 60°C. For each sample the level of target gene transcripts was normalized to β-actin.