Quantitative real-time PCR

Total RNA gathered from snap-frozen tissue samples was isolated using Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The gene-specific primers for CD155 were designed as follows: forward, 5′-TATCTGGCTCCGAGTGCTTGCC-3′ and reverse, 5′-ACGACGGCTGCAAAAGTGGCG-3′. Glyceraldehyde 3-phosphate dehydrogenase was used as the internalized control, and the sequences were shown as follows: forward: 5′-CCTAGTTCGTCATGGGTGTGAACCA-3′ and reverse: 5′-GCCAGTAGAGGCAGGGATGATGTTC-3′. CD155 level was determined by SYBR Green-based RT-PCR performed on a PikoReal RT-PCR system (Thermo Fisher Scientific, Waltham, MA, USA) in the following conditions: an initial denaturation step for 10 minutes at 95°C, followed by 40 amplification cycles involving denaturation for 15 seconds at 95°C, then annealing for 30 seconds at 60°C, and finally elongation for 30 seconds at 72°C. We performed melting-curve analysis to monitor PCR product purity, and the data of relative gene expression were analyzed using the 2^−ΔΔCt method.