Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from the tumor tissues of 45 patients from whom fresh tissues were available using RNA extraction buffer from the Promega SV Total RNA Isolation System kit, (cat. no. Z3100; Promega Corporation) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using the cDNA synthesis kit Roche Transcriptor cDNA Synth. Kit 2 (cat. no. 4897030001; Roche Diagnostics) according to the manufacturer's instructions (first step 65°C for 10 min, then 65°C for 30 min and finally 85°C for 5 min). qPCR was subsequently performed using SYBR Master Mix (Roche Diagnostics) on a LightCycler 480 Real Time PCR system (Roche Diagnostics). The thermocycling conditions were as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and 72°C for 1 min with a final extension step at 72°C for 10 min. The primers used for RT-qPCR were as follows: PD-L1, forward: 5′-GTACCGCTGCATGATCAGCTAT-3′ and reverse: 5′-GGCATTGACTTTCACAGTAATTCG-3′; IFN-γ, forward: 5′-TCTGGATCCATGAACGCTACACACTGC-3′ and reverse: 5′-ACTAAGCTTTCAGCAGCGACTCCTTTTCC-3′; JAK2, forward: 5′-CTGCAGGAAGGAGAGAGGAAGAGGA-3′ and reverse: 5′-GAATGTTATTGGCAGTCAG-3′; STAT1, forward: 5′-CCACTGAGACATCCTGCCACC-3′ and reverse: 5′-CCACTGAGACATCCTGCCACC-3′ and GAPDH, forward: 5′-ACCACAGTCCATGCCATCACT-3′ and reverse: 5′-ACTGTGCCGTTGAATTTGCC-3′. GAPDH was used as the endogenous reference gene. Expression levels of PD-L1, IFNG, JAK2 and STAT1 were quantified using the 2^−ΔΔCq method (23).