Itaq fast sybr green supermix with rox

Total RNA was extracted with GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich), and 1 μg RNA was reverse-transcribed to cDNA with the iScript cDNA Synthesis Kit (Bio-Rad). An ABI 7500 real-time PCR instrument (Applied Biosystems) was used to perform quantitative PCR (qPCR) analysis, using iTaq Fast SYBR Green Supermix with ROX (Bio-Rad) in 96-well plates at a final reaction volume of 10 μL. The qPCR analysis was carried out in triplicate with the following primer sets: HSD3B1, 5′-CCATGTGGTTTGCTGTTACCAA-3′ (forward), 5′-TCAAAACGACCCTCAAGTTAAAAGA-3′ (reverse); PSA, 5′-GCATGGGATGGGGATGAAGTAAG-3′ (forward), 5′-CATCAAATCTGAGGGTTGTCTGGA-3′ (reverse); FKBP5, 5′-CCCCCTGGTGAACCATAATACA-3′ (forward), 5′-AAAAGGCCACCTAGCTTTTTGC-3′ (reverse); TMPRSS2, 5′-CCATTTGCAGGATCTGTCTG-3′ (forward), 5′-GGATGTGTCTTGGGGAGCAA-3′ (reverse); RPLP0 (large ribosomal protein P0, a housekeeping gene), 5′-CGAGGGCACCTGGAAAAC-3′ (forward, 5′-CACATTCCCCCGGATATGA-3′ (reverse).
For steroid-treated cells, each mRNA transcript was quantitated by normalizing the sample values to RPLP0 and to vehicle-treated cells. All gene expression studies were repeated in at least 3 independent experiments.

RNA was extracted from A549 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). An iScript reverse transcription supermix kit (Bio-Rad, Hercules, CA, USA) was used for reverse transcription. PCR amplification mixtures were prepared using iTaq™ Fast SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA, USA). The sequences of the primers for ezrin were as follows: forward 5′-GTG GGA TGC TCA AAG ATA ATG C-3; reverse 5′-CAC CTC GAT GGT GTC AGG CT-3′. Real-time PCR was performed using an Mx3000p system (Stratagene, La Jolla, CA, USA) and all samples were run in triplicate. The quantification of gene expression levels was calculated relative to β-actin.