Rg 6000 thermocycler

Tumour content of RNA was extracted using Trizol (GeneAll, Korea), which was subjected to quality and quantity assessments using agarose gel visualization and measuring the A260/A280 ratio (NanoDrop 1000 Spectrophotometer, Wilmington, DE, USA). Complementary DNA was generated using Two‐Strand Synthesis kit (General, Korea), and gene expression analysis was performed using real‐time PCR. Each PCR cycle included a 10‐s denaturation phase at 95°C, followed by a 10‐s annealing step at 55–62°C and a 30‐s extension phase at 72°C. Each PCR reaction contained template cDNA (1 μL), specific pairs of primers (1 μL), deionized water (10.5 μL) and SYBR® Green Master Mix‐Plus (Yektatajhiz, Iran) (12.5 μL) performed in Corbett RG6000 thermocycler (Australia). GAPDH was employed as the internal control gene, and the Rotor‐Gene 6000 Series Software was used to calculate the cycling threshold (CT) (Table 1). The CT (Cq) obtained was adjusted according to the respective value obtained for the internal control gene using the following formula: Cq^target − Cq^GAPDH = ΔCq. Finally, 2^(−ΔΔCq) formula was used to calculate gene expression in the sample (Livak & Schmittgen, 2001), where ΔΔCq = ΔCq^target – ΔCq^control.