Total RNA (0.5 μg), which was isolated from the bladder tissue specimens, using Isogen (NipponGene, Tokyo, Japan), was reverse transcribed using 1 μM oligo (dT) primers (Qiagen, Germantown, MD, USA) and 4 units of Omniscript reverse transcriptase (Qiagen, Germantown, MD, USA) in a total volume of 20 μL. Real-time quantitative PCR (qPCR) was then performed (StepOne Real Time PCR System, Applied Biosystems, Grand Island, NY, USA), using Fast SYBR Green Mastermix (Applied Biosystems, Grand Island, NY, USA), as described previously (22086872) [
19] The following primer pairs were used for the RT-PCR: human
PD-L1: forward 5′- CCA AGG CGC AGA TCA AAG AGA’; reverse 5′- AGG ACC CAG ACT AGC AGC A -3′, and human
NFATc1: forward 5′- GTC CCA CCA CCG AGC CCA CTA CG -3′; reverse 5′- GAC CAT CTT CTT CCC GCCC ACG AC -3′. Human
GAPDH: forward 5’-CTC CTC CAC CTT TGA CGC TG-3′; reverse, 5’-CAT ACC AGG AAA TGA GCT TGA CAA-3′ was used as an internal control. The sequences of these primers were acquired from the Primer Bank: 19906719 [
20], 19,108,745 [
21], 14,654,707 [
22]. The
PD-1 and
STAT1 gene expressions were determined using TaqMan® Gene Expression Assays (
PD-L1 and
STAT1, Applied Biosystems, Grand Island, NY, USA). All of the specific expression levels were divided by the quantity of
GAPDH that was used.