Abi 7500 real time pcr detection system

Total DNA and RNA from kidney cortex tissues were isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA) and TRIzol reagent (TaKaRa), respectively. Oligonucleotides were designed using Primer5 software (available at http://frodo.wi.mit.edu/) and synthesized by Invitrogen. Reverse transcription was performed using the Promega Reverse Transcription System according to the manufacturer's protocol (Madison, WI). Real-time PCR amplification was performed using the ABI 7500 Real-Time PCR Detection System (Foster City, CA) by using SYBR Premix Ex Taq (TaKaRa). The cycling program consisted of a preliminary denaturation (95°C for 10 min), followed by 40 cycles (95°C for 15 s and 60°C for 1 min). The relative mitochondrial DNA copy number was normalized to the 18S rRNA level encoded by the nuclear DNA, and mRNA levels were normalized to GAPDH and calculated using the comparative cycle threshold (ΔΔCt) method. The primer sequences were shown in Table 1.

Total RNA was from cultured MCs by using a TRIzol reagent (TaKaRa) according to the manufacturer's protocol. Reverse transcription was performed using a PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's protocol. Oligonucleotides (cyclin D1: forward, 5′-CGC CCT CCG TTT CTT ACT TC-3′, and reverse, 5′-GCA GTC AGG GGA ATG GTC T-3′; cyclin A2: forward, 5′-AAG ATG CCC TGG CTT TTA GTG-3′, and reverse, 5′-TAACATTCACTGGCTTTTCGTCT-3′; Cyclooxygenase-2: forward, 5′-AGGACTCTGCTCACGAAGGA-3′, and reverse, 5′-TGACATGGATTGGAACAGCA-3′; and GAPDH: forward, 5′-GTCTTCACTACCATGGAGAAGG-3′, and reverse, 5′-TCATGGATGACCTTGGCCAG-3′) were designed using Primer 5 software (available at http://frodo.wi.mit.edu/) and synthesized by Invitrogen. Real-time PCR amplification was performed using the ABI 7500 Real-Time PCR Detection System (Foster City, CA) by using SYBR Premix Ex Taq (TaKaRa). The cycling program consisted of a preliminary denaturation (95°C for 10 min), followed by 40 cycles (95°C for 15 s and 60°C for 1 min). Relative gene expression of mRNA was normalized to GAPDH and calculated using the ΔΔCt method.

Total cellular RNA was extracted using RNAiso plus reagent (9,108, Takara, Kyoto, Japan) according to the manufacturer’s instructions. Subsequently, the total RNA concentration was determined with a NanoDrop 2.0 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, United States) and the RNA was reverse transcribed to cDNA using a PrimeScript™ RT reagent kit with gDNA Eraser (RR047A, Takara) according to the manufacturer’s instructions. Subsequently, qRT-PCR assays were performed by using a SYBR Premix Ex Taq™ II (2×) kit (RR820A, Takara) according to the manufacturer’s instructions and run on an ABI 7500 Real-Time PCR Detection System (Foster City, CA, United States). The reactions were performed using the following parameters: 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The primer nucleotide sequences used for qRT-PCR are listed in Supplementary Table S1. All primer sets for mRNA amplification were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The relative expression levels of the target gene were normalized with respect to the levels of β-actin expression and calculated using the 2^−△△CT method.