RNA Extraction and qRT-PCR for Gene Expression

Kidney tissue samples were dissected, snap-frozen in liquid N2, and kept at -80°C until use. Total tissue RNA was extracted using a mirVana miRNA Isolation Kit (Thermo Fisher Scientific). BUMPT cell RNA was extracted using a GeneJET RNA Purification Kit (Thermo Fisher Scientific). To measure Tnfa mRNA, 2 μg RNA was reversely transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and real-time PCR (RT-PCR) was performed using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific). Gapdh was used as an internal control. For the other mRNAs, 1 μg RNA was reversely transcribed using a cDNA Transcription Kit (Bio-Rad), and RT-PCR was performed using SYBR Green PCR Master Mix (Bio-Rad). β-Actin was used for normalization. The quantification was done using ΔCt values as recently described (45 (link), 46 (link)). Predesigned RT-PCR probes were used for mouse Tnfa and mouse Gapdh. The sequences of the other primers were shown in Table S1. All primers were synthesized at Integrated DNA Technologies Inc.