I cycler iqtm5 detection system

Real-time RT-PCR analysis was performed by using gene-specific primers (Table 1, MWG), SYBR Green PCR Mastermix (Qiagen) and the I-Cycler IQ^TM5 detection system (Bio-Rad, Munich, Germany). Standard and melt curves were performed to determine PCR efficiency and specificity of amplification, respectively. Mean cycle thresholds (Ct) values were normalized to the reference gene β2-microglobulin (βMG). Neuroblastoma cell line SH-SY5Y or colon carcinoma cell line CaCo-2 were used as positive controls.

Total RNA was extracted from isolated renal cortex using the RNAprep Pure Tissue Kit (TianGen, China) following the manufacturer’s instructions. First-strand cDNA was obtained by reverse transcription of the total RNA using the TIANscript RT Kit (TianGen, China). The resulting cDNA and corresponding primers were used for SsoFast^TM Eva Green Supermix-quantitative real-time PCR to assay levels of MCP-1, ICAM-1, IL-6, IL-1β, PDGF-BB, Col I, Col IV, FN-1, and TGF-β₁. The primers were designed based on the mRNA sequences in GenBank and synthesized by Shanghai Shenggong Biotechnology (Shanghai, China). Quantitative QT-PCR was carried out on an iCycler iQ^TM5 detection system (Bio-Rad, USA) with rat β-actin as internal control. Optimal PCR conditions and primers are summarized in Supplementary Table 3. The normalized fold expressions of tested gene relative to the normal control was calculated based on the 2^−ΔΔCt method^{61 (link)}, where Ct is the mean threshold cycle difference.