Quantitative DNA Methylation Analysis of CYP11B1 Promoter

Genomic DNA was extracted from CPA, AUAT, normal ZF, and NFT flash-frozen (CPA n = 12, AUAT n = 12, normal ZF n = 4, NFT n = 4) or formalin-fixed paraffin-embedded tissue (CPA of Case 10, AUAT of Case 12, 3 normal ZF, and 3 NFT which could not be obtained as flash-frozen) using Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany). We collected normal ZF samples from normal adrenal cortex with flash-frozen (n = 4) or formalin-fixed paraffin-embedded tissue (n = 3). The specimens from seven patients with NFT (n = 4), renal cell carcinoma (n = 1), and extra-adrenal paraganglioma (n = 2) were examined. We scraped the normal ZF area to extract DNA as previously reported^{28 (link)}. Genomic DNA from WBC was extracted using Puregene Blood Core Kit (Qiagen). Genomic DNA from samples was treated with bisulfite and PCR amplified with primers specific for human CYP11B1 promoter regions (Table S1). Quantitative methylation analysis of the PCR products was performed with PyroMarkGold Q96 Reagents and the PyroMarkQ24 pyrosequencing system (Qiagen).