Icycler iq single color real time pcr detection system

Total RNA was extracted using the InviTrap® Spin Cell RNA Mini Kit (INVITE). RNA purity was checked using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized by using 1^st Strand cDNA Synthesis Kit for RT-PCR (AMV) and RT-qPCR was performed with Sybr green IQ Supermix (Bio-Rad Laboratories), using an iCycler iQ single-color real-time PCR detection system (Bio-Rad Laboratories). Fold changes in expression versus control condition were determined using the 2^(−ΔΔCt) method^{70 (link)} with 16s-rRNA as a housekeeping gene (see Supplementary Table 6 for primers sequence).

Total RNA was isolated from HME-1 cells treated with different concentrations (0, 5 and 20 nM) of 1,25(OH)₂D for 24 h using RNAzol reagent (Molecular Research Center, Inc. Cincinnati, OH). The reverse transcription reaction was performed using poly-dT primer and moloney murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) in a 25 μl reaction volume containing total RNA (2 μg), 1× PCR buffer and 2 mM MgCl₂, at 42 °C for 15 min followed by 95 °C for 5 min. The quantitative real-time RT-PCR was performed using IQ^™ SYBR Green Supermix in an iCycler (iCycler iQ Single-color real-time-PCR detection system; Bio-Rad, Hercules, CA). The primer sequences used to amplify human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) mRNA were sense 5′-CCT ACC CCC AAT GTA TCC GTT GTG-3 and anti-sense 5′-GGA GGA ATG GGA GTT GCT GTT GAA-3′; TRAIL mRNA primer sequences were sense 5′-TTC ACA GTG CTC CTG CAG TC-3′ and antisense 5′-CAG CAG GGG CTG TTC ATA CT-3′. Thermal cycling parameters were 94 °C for 4 min, followed by 35 cycles of amplifications at 94 °C for 30 s, 58 °C for 1 min, 72 °C for 2 min, and 72 °C for 10 min as the final elongation step. Relative levels of mRNA expression were normalized in all the samples analyzed on the levels of GAPDH amplification.