Quantitative Gene Expression Analysis

Total RNA was isolated from 1 × 10⁶ cells using the RNeasy Plus Mini Kit (Qiagen) per manufacturer's instructions. The concentration and purity of the recovered RNA was determined measuring the optical density at 260 and 280 nm. cDNA was generated using SuperScript™II Reverse Transcriptase (Thermo Fisher Scientific Inc.) per the manufacturer's instructions. For qPCR, 5 μL of Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific Inc.), 5 μL of 1:25 diluted cDNA and 500 nM of each primer of interest was used in a 10 μL reaction. The reaction was performed using a 7900 Real-Time PCR System (Thermo Fisher Scientific Inc.). The samples were run at 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 94°C for 15 seconds, 58°C for 35 seconds, and 72°C for 35 seconds followed by a standard dissociation stage to determine the melting temperature of each amplification product. The comparative quantitation method was used for data analysis. The results were presented as expression fold relative to GAPDH : 2^{−(C_t target gene-C_tGAPDH}). Primers used in the study were described previously (10 (link)). Results shown are representative of three independent experiments.