RASSF1A expression and regulation in adrenal tumor. (A) Averages of percentage methylated (FM) and unmethylated (FUM) CpGs in CpG island A of RASSF1A promoters in Normal adrenal cortex (n = 6), ACA (n = 8), and ACC (n = 7) samples are shown. FM includes both Hypermethylated (FHM) and intermediate methylated (FIM) fractions. (B) Methylation profiles of individual fresh-frozen normal adrenal cortex (N1 – N6), 8 ACAs (A1 – A8) and 7 ACC samples (C1 – C7) as determined by Epitect methyl II PCR assay. (C) Expression of RASSF1A mRNA determined by real-time qPCR in 7 ACC samples (C1 – C7) compared to the average expression in 6 normal samples (N) normalized to a value of 1.0. RASSF1A expressions in individual samples were also normalized to the average mRNA expression of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP). C-Av represents the average expression of all ACC samples. Data shown is from one of triplicate experiments that yielded similar results (mean ± SD). Independent sample t-test used to derive the p value (p = <0.01). (D) RASSF1A protein expression in normal (a &b) and ACC (c &d) FFPE tissue specimens demonstrated by immunohistochemistry through DAB staining (brown indicates RASSF1A protein expression) followed by nuclear counterstaining by hematoxylin (blue).