Total RNA was isolated from bladder epithelial cells using E.Z.N.A. ® Total RNA Kit I (Omega Bio-tek, GA, USA) according to manufacturer's protocol. The concentration of RNA was determined using spectrophotometry (Nano- Drop ND-1000, Wilmington, NC, USA). First strand cDNA synthesis was performed by High capacity cDNA RT kit (Thermo Fisher Scientific). The real time-RT-PCR was carried out in a total volume of 10 μl on a 96-well plate (Sarstedt, Nümbrecht, Germany). Each well contained 5 μl Maxima SYBR Green qPCR Master Mix (Thermofisher), 1 μl of each primer IL-1β (Forward: 5′-CCACAGACCTTCCAGGAGAATG-3, Revers: 5′-GTGCAGTTCAGTGATCGTACAGG-3′), NLRP3 (Forward: 5′-GGACTGAAGCACCTGTTGTGCA-3, Revers: 5′-TCCTGAGTCTCCCAAGGCATTC-3′), pro-caspase-1 (Forward: 5′-GCTGAGGTTGACATCACAGGCA-3, Revers: 5′-TGCTGTCAGAGGTCTTGTGCTC-3′), ASC (Forward: 5′-AGCTCACCGCTAACGTGCTGC-3, Revers: 5′-GCTTGGCTGCCGACTGAGGAG-3′), uroplakin 1a (Forward: 5′-CACCAAGCAGATGCTGACCTTC−3, Revers: 5′-GGACCAGATGTGCCACAGCATT-3′), uroplakin 3a (Forward: 5′-CTCACAGATCCTGAATGCCTACC-3, Revers: 5′-CCGTGGACATATTGACCAGGAC-3′), integrin α3 (Forward: 5′-GCCTGACAACAAGTGTGAGAGC-3, Revers: 5′-GGTGTTCGTCACGTTGATGCTC-3′), integrin β1 (Forward: 5′-GGATTCTCCAGAAGGTGGTTTCG-3, Revers: 5′-TGCCACCAAGTTTCCCATCTCC-3′), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Forward: 5′-GTCTCCTCTGACTTCAACAGCG-3, Revers: 5′-ACCACCCTGTTGCTGTAGCCAA-3′) (Eurofins MWG Synthesis GmbH, Ebersberg, Munich), 1 μl cDNA (10 ng) and 3 μl RNase free water. The PCR amplification was conducted in a CFX96 Touch™ Real-Time PCR Detection System (Biorad, Hercules, CA, USA) using the following protocol: initial denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s followed by annealing/extension at 60°C for 60 s. Each PCR was followed by a dissociation curve analysis between 60-95°C. The Ct values were analyzed by the comparative Ct (ΔΔCt) method and normalized to the endogenous control GAPDH. Fold difference was calculated as 2−ΔΔCt.
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