The methylation data were processed using the Bioconductor lumi package. The methylation data was color balance check, background corrected, and normalized using quantile normalization, based on the assumption that the intensity distribution of the pooled methylated and unmethylated probes are similar for different samples . 89 CpG sites with more than 25% of samples having detection p-values worse than 0.05 were filtered before the analysis. Methylation values for each CpG locus are expressed as a ﾓM-valueﾔ, calculated as the log2 ratio of the intensities of methylated probe versus unmethylated probe for a given locus,
Microarrays were washed under high stringency, labeled with biotin (C and G nucleotides) or dinitrophenyl (A and T nucleotides), and scanned with an Illumina iScan.
DNA was hybridized to Illumina Infinium HumanMethylation27 arrays, containing 50-mer oligonucleotides designed to hybridize either methylated (N-CG-N following bisulfite conversion) or unmethylated (N-TG-N following bisulfite conversion) cytosine on each CG pair interrogated, coupled to beads mounted on glass slides.
Bisulfite-converted DNA samples were prepared and quantified using a NanoDrop ND-1000 spectrophotometer. For each sample, 500ng of whole-genome bisulfite-converted DNA was denatured, fragmented, and amplified using Illumina-supplied reagents according to manufacturer instructions. DNA samples were not labelled because after hybridization, allele-specific single-base extension incorporates fluorescent labels for detection.
Tumor samples (10 to 50 mg) were powdered under liquid nitrogen. DNA was extracted and purified by cesium chloride gradient ultracentrifugation (30) or by proteinase K digestion and ethanol extraction, followed by a clean-up step on columns (Qiagen, Courtaboeuf, France).
The tumors were prospectively collected in the tumor bank of the COMETE network (Cortico-Medulo Tumeurs Endocrines). One hundred thirthy-five tumors, collected between 1993 and 2005 by the Cochin team, were included in this study. Samples were dissected by the pathologist immediately after tumor removal, snap frozen and kept in liquid nitrogen.