Quantitative PCR analysis of Nephrin gene

Total RNA from CIHP-1 cells seeded into a 6-well plate (6x10⁴ cells/well) was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the PrimerScript reverse transcriptase (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. qPCR analysis was performed on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR green Premix Ex Taq II (Qiagen GmbH), according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 5 min; followed by 40 cycles of denaturation at 95 ˚C for 45 sec, annealing at 50˚C for 45 sec and elongation at 72˚C for 45 sec; and a final extension step at 72˚C for 10 min. GAPDH was used as an internal reference gene and the relative gene expression was determined using the 2^-ΔΔCq method (24 (link)). The following primer pairs were used for qPCR: Nephrin forward, 5'-CTGCCTGAAAACCTGACGGT-3' and reverse, 5'-GACCTGGCACTCATACTCCG-3'; and GAPDH forward, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse, 5'-ACACCATGTATTCCGGGTCAAT-3'.