tissue type: Breast carcinoma histological assessment: Tubular carcinoma
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted according to the Tissue Protocol of the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). After incubation with AL buffer an additional centrifugation step (2000 x g, 10 min) was added for fat removal. In Methyl-CpG Immunoprecipitation (MCIp) two µg ultrasonicated DNA (with an average fragment size of 400-500 bp) were immunoprecipitated with 60 µg MBD-Fc protein. MDB-Fc protein was produced according to a protocol described by Gebhard et al. (Cancer Res, 2006, 66 (12): p. 6118-28). Unbound DNA was recovered by centrifugation, bound DNA eluted twice with buffers containing increasing NaCl concentrations (400, 500, 550, 600 and 1000 mM NaCl). Eluates were desalted and fractions containing highly methylated DNA (600 and 1000 mM fractions) were combined for each sample.
Label
Alexa Fluor 3
Label protocol
Samples were labelled with (BioPrime Total Genomic Labeling System, Invitrogen): tumor with Alexa Fluor 3 and normal with Alexa Fluor 5.
tissue type: Normal breast histological assessment: Normal breast tissue
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted according to the Tissue Protocol of the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). After incubation with AL buffer an additional centrifugation step (2000 x g, 10 min) was added for fat removal. In Methyl-CpG Immunoprecipitation (MCIp) two µg ultrasonicated DNA (with an average fragment size of 400-500 bp) were immunoprecipitated with 60 µg MBD-Fc protein. MDB-Fc protein was produced according to a protocol described by Gebhard et al. (Cancer Res, 2006, 66 (12): p. 6118-28). Unbound DNA was recovered by centrifugation, bound DNA eluted twice with buffers containing increasing NaCl concentrations (400, 500, 550, 600 and 1000 mM NaCl). Eluates were desalted and fractions containing highly methylated DNA (600 and 1000 mM fractions) were combined for each sample.
Label
Alexa Fluor 5
Label protocol
Samples were labelled with (BioPrime Total Genomic Labeling System, Invitrogen): tumor with Alexa Fluor 3 and normal with Alexa Fluor 5.
Hybridization protocol
Tumor and normal samples were paired randomly (except for Array 10 where matching normal tissue was available). Samples were hybridized to Human CpG Island microarrays (Agilent Technologies, Santa Clara, CA, USA) using the Oligo aCGH Hybridization Kit (Agilent). Array hybridization and washing was carried out according to the Agilent Microarray Analysis of Methylated DNA Immunoprecipitation protocol (Version 1.0, May 2008; Agilent Technologies).
Scan protocol
Arrays were scanned with Agilent Technologies Scanner G2505B US22502640.
Data processing
Micorarray data were extracted with Agilent Feature Extraction Software 10.5 using CGH_105_Dec08 protocol. Since normal tissue was detected in red channel and tumor tissue in green channel, in order to obtain LogRatio(Tumor/Normal) the LogRatio values from original extraction files were multiplied by -1. Array data were corrected for GC content as reported previously by Schilling et al. (Genome Res, 2009, 19(11): p. 2028-35).
