High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis Marija.

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High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis Marija Balic, Martin Pichler, Jasmin Strutz, Ellen Heitzer, Christoph Ausch, Hellmut Samonigg, Richard J. Cote, Nadia Dandachi The Journal of Molecular Diagnostics Volume 11, Issue 2, Pages 102-108 (March 2009) DOI: 10.2353/jmoldx.2009.080109 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Sensitivity of HRM assays for MGMT (A) and APC (B) methylation. Assays were run at annealing temperatures ranging from 64°C to 58°C using a touchdown protocol. Data were analyzed using software modules “Gene Scanning” and “Difference Plot.” Methylation standards are displayed as triplicates. Standards 100% blue lines, 50% red lines, 25% green lines, 10% pink lines, 1% yellow lines, 0.1% brown lines, and 0% orange lines. The Journal of Molecular Diagnostics 2009 11, 102-108DOI: (10.2353/jmoldx.2009.080109) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Reproducibility analysis of the APC HRM assay. A: Interassay variability for the APC HRM assay. The same set of bisulfite-converted methylation standards were subjected to four independent HRM assays performed on different days. B: Influence of bisulfite treatment on methylation results. The bisulfite treatment was performed on four different days and bisulfite-treated methylation standards were subjected to the same APC HRM assay. The Journal of Molecular Diagnostics 2009 11, 102-108DOI: (10.2353/jmoldx.2009.080109) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Interassay variability for the APC HRM assay (A) and MethyLight assay (B) using fresh and FFPE cancer cell line DNA from eight breast and prostate cancer cell lines. The bars represent the mean of six independent experiments and the SD. The Journal of Molecular Diagnostics 2009 11, 102-108DOI: (10.2353/jmoldx.2009.080109) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 A: APC HRM curves for methylation standards containing varying amounts of methylated DNA (colored lines) and four samples (three methylated and one unmethylated) represented by black lines. B: Difference plot for the data represented in (A). Melting curves were normalized to the 0% standard. Methylation standards and samples are displayed as triplicates. Standards 100% blue lines, 50% red lines, 25% green lines, 10% pink lines, 1% yellow lines, 0.1% brown lines, and 0% orange lines. The Journal of Molecular Diagnostics 2009 11, 102-108DOI: (10.2353/jmoldx.2009.080109) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions