Real-Time Quantitative PCR Analysis

mRNA expression levels of collagen IV, fibronectin, p62, Beclin-1, mTOR, and ULK1 in kidneys were detected by real-time quantitative PCR. According to the manufacturers’ protocols, total RNA was extracted from kidney tissue using TRIzol and cDNA was synthesized by reverse transcription, cDNA and TB Green Premix Ex Taq II were premixed for PCR. The thermal reaction cycle was as follows: 95°C for 30 s, and then 40 cycles of 95°C for 5 s and 60°C for 30 s (CFX Connect, Bio-Rad, United States). β-actin was used as a housekeeping gene to standardize Ct values. Fold changes of mRNA expression were calculated by relative quantification (2^−ΔΔCt). Primer sequences used for PCR are shown in Table 2.

Primer sequences used for PCR.