Brilliant 2 qrt pcr master mix

Total RNA was extracted from cells using the RNeasy Plus Mini kit (Qiagen, Germantown, MD). The quantity and quality of RNA was confirmed by absorption measurements at 260 and 280 nm. Single strand cDNA synthesis and PCR amplification were carried out in a 1-step reaction using the Brilliant II QRT-PCR Master Mix (Agilent, Santa Clara, CA). 100 ng of RNA was loaded in 96-well plate format and amplified with 0.9 μM of primers, 0.25 μM of MGB probe, and 30 nM of ROX passive reference dye. The reaction was carried out in a Mx3005P instrument (Agilent) with the following parameters: an RT step at 50°C for 30 min was followed by a preincubation step at 95°C for 10 min, 50 cycles of denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min. Efficiencies for each primer/probe set were calculated from a serial dilution of Human Reference Total RNA (Agilent) and were >95%. Relative quantification of gene expression was calculated with the comparative cycle threshold method, normalizing for GAPDH expression levels.