Cfx96 real time system thermocycler

RNA was isolated from 29 fresh frozen ACC samples, 10 normal adrenal samples, and two human ACC cell lines (NCI-H295R and SW-13) using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). This cohort included all 27 samples accrued for the CNV analysis and 2 additional ACC tumors acquired at a later date. Quantity and quality of isolated RNA was assessed by spectrophotometry (NanoDrop Technologies, Wilmington, DE). The synthesis of cDNA was performed with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) using 200 ng of RNA per sample. Quantitative RT-PCR was performed in a CFX96 Real-Time System thermocycler (Bio-Rad) using TaqMan PCR master mix with primers and probes specific to the CYP genes CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2S1, & CYP4F2 (all from Applied Biosystems). Relative expression between samples was normalized to that of endogenous ribosomal protein, large, P0 (RPLP0) expression. The presented data are representative of two independent experiments performed with duplicate samples for all assays. Relative mRNA expression levels were calculated by first normalizing the mean expression in the non-tumor adrenal samples to 1.0. This arbitrary value was then compared to the expression level in tumor samples and fold-change was calculated using the Livak method.^{18 (link)}