High-molecular weight DNA was isolated from fresh-frozen normal and tumor tissue samples using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Methyl-CpG binding domain (MBD)-Fc protein was produced and MCIp performed as described previously [Schilling, Genomics, 2007] with minor modifications. Briefly, 60 µg of MBD-Fc protein was coupled to 150 µl nProtein A Sepharose beads (GE Healthcare, Neu Isenburg, Germany) at 4°C overnight. After binding of 2 µg sonicated DNA mixed with 2 pg of a mixture of in-vitro methylated and unmethylated synthetic DNA of 630 bp containing 47 CpGs, DNA elution was performed with increasing NaCl concentrations (300-1000 mM) using Ultra-free-CL centrifugal filter devices (Merck-Millipore, Billerica, USA). Low and high salt concentrations were used to enrich for lowly and highly methylated DNA, respectively.
Label
Alexa Fluor 5
Label protocol
Highly methylated tumor and normal mucosa DNAs were labeled with Alexa Fluor® 5 and 3 (BioPrime Total Genomic Labeling System, Invitrogen, Karlsruhe, Germany), respectively, following the instructions of the manufacturer.
High-molecular weight DNA was isolated from fresh-frozen normal and tumor tissue samples using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Methyl-CpG binding domain (MBD)-Fc protein was produced and MCIp performed as described previously [Schilling, Genomics, 2007] with minor modifications. Briefly, 60 µg of MBD-Fc protein was coupled to 150 µl nProtein A Sepharose beads (GE Healthcare, Neu Isenburg, Germany) at 4°C overnight. After binding of 2 µg sonicated DNA mixed with 2 pg of a mixture of in-vitro methylated and unmethylated synthetic DNA of 630 bp containing 47 CpGs, DNA elution was performed with increasing NaCl concentrations (300-1000 mM) using Ultra-free-CL centrifugal filter devices (Merck-Millipore, Billerica, USA). Low and high salt concentrations were used to enrich for lowly and highly methylated DNA, respectively.
Label
Alexa Fluor 3
Label protocol
Highly methylated tumor and normal mucosa DNAs were labeled with Alexa Fluor® 5 and 3 (BioPrime Total Genomic Labeling System, Invitrogen, Karlsruhe, Germany), respectively, following the instructions of the manufacturer.
Hybridization protocol
Each tumor sample was cohybridized with the matched normal mucosa on a 244K Human CpG Island microarray using the Oligo aCGH/ChIP-on-chip Hybridization Kit (Agilent technologies, Palo Alto, USA), following the instructions of the manufacturer.
Scan protocol
Arrays were scanned on an Agilent G2505B scanner.
Data processing
Images were quantified using Agilent Feature Extraction Software (version A.10.5.1.1). Scanned raw data were processed using the R statistical environment. Background correction and log 2-ratio transformation were performed according to the NormExp method with offset = 50 [Ritchie, Bioinformatics, 2007]. To reduce variations between co-hybridized samples, intensity-based LOESS normalization on rank-invariant probes and negative controls was applied [Tseng, Nucleic Acids Res, 2001]. For between-array normalization, log-intensity ratios (M-values) and log-intensity averages (A-values) were scaled to have the same median-absolute-value across the arrays.
