2.8 Real-time RT-PCR

To quantify the expression of CYP24A1 mRNA and its induction by 1,25D3, cells were treated with EtOH or 10⁻⁷ M 1,25D3 for 24 hours. Total RNA was isolated using Trizol (Life Technologies, Grand Island, NY) according to manufacturer’s protocols. cDNA was prepared in a 20 μl reaction from 1 μg of total RNA by cDNA iScript^™ cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR reactions were run in triplicates (n=3) in the iCycler iQ^™ real-time PCR detection system (Bio-Rad, Hercules, CA) in a 20 μl reaction mixture containing 2 μl of the reverse transcription product, 0.75 μl of 10 μM stocks of forward and reverse primers [36], and 10 μl of iTaq Universal SYBR Green Supermix. Reactions were run with the following parameters: 95°C for 3 minutes followed by 40 cycles of 95°C for 10 seconds, 55°C for 1 minute and GAPDH was run as a control for CYP24A1 expression. ΔCt values and fold of induction were calculated as described previously [12]. Each data point represents three independent analyses (n=3) presented as mean ± SEM. *p<0.05, ***<0.0005, ****p<0.0001.