Taqman primers

Total RNA was extracted from the kidney cortex using an RNAeasy mini kit ( Cat# 74104, Qiagen, Hilden, Germany) and used for cDNA synthesis by using RT2 First Strand Kit (Cat# 330404, Qiagen, Hilden, Germany). The polymerase chain reaction for NQO-1 and β-actin was performed with commercially available TaqMan primers (Integrated DNA Technologies, Coralville, IA). The following sequences of the primers were used: NQO-1 (forward: 5’-CCATGTACTCTCTTCAGG-3’ and reverse:5’-GCCAATGCTGTAAACCAGTTG-3’) and β-actin (forward:5’-GATTACTGCTCTGGCTCCTAG-3’ and reverse: 5’- GACTCATCGTACTCTCCTGCTTG-3’).
Immunoblotting in the renal cortical homogenates was performed using the standard lab method as detailed previously (22 (link)). Briefly, tissue homogenates were centrifuged at 12000 rpm for 10 minutes at 4 °C and the supernatant was solubilized in Laemmli buffer, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes at 4°C. The membranes were blocked with 5% nonfat dry milk and incubated with antibodies against Nrf2 (1:500, Cat# 16396-1-AP, Proteintech) and NQO-1 (1:500, Cat# bsm-52830R, Bioss) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000, Cat# 2118, Cell Signaling) followed by corresponding horseradish peroxidase conjugated secondary antibodies.

Total RNA was extracted from tissue or cells using Trizol reagent (Invitrogen, Thermo Fisher Scientific) followed by reverse transcription for cDNA synthesis from mRNA or miRNA using iScript^™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and TaqMan® MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific), respectively. The miRNA levels were determined with TaqMan® primers (Integrated DNA Technologies, Coralville, IA, USA) as indicated by quantitative real-time PCR using TaqMan® Fast Universal PCR Master Mix (Thermo Fisher Scientific). All qPCR reactions were run using the Applied Biosystems ABI7900HT PCR machine.