Accurate Detection of Copy Number Changes in DNA Extracted from Formalin-Fixed, Paraffin-Embedded Melanoma Tissue Using Duplex Ratio Tests David A. Moore,

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Accurate Detection of Copy Number Changes in DNA Extracted from Formalin-Fixed, Paraffin-Embedded Melanoma Tissue Using Duplex Ratio Tests David A. Moore, Gerald Saldanha, Abdlrzag Ehdode, Linda Potter, Lovesh Dyall, Danielle Bury, J. Howard Pringle The Journal of Molecular Diagnostics Volume 15, Issue 5, Pages 687-694 (September 2013) DOI: 10.1016/j.jmoldx.2013.04.008 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Comparison of diploid tonsil DNA versus melanoma cell-line DNA for the BRAF versus PTEN DRT assay. In the diploid sample, the equal quantity of DNA for the two target genes is reflected in overlapping amplification plots for each product and identical CT values for BRAF and PTEN. In the melanoma sample, the DNA-gain target (BRAF) has a lower CT than the DNA-loss target (PTEN), indicating a difference in DNA copy number between the two targets for this DRT. The difference between the two CT values is the ΔCT. The Journal of Molecular Diagnostics 2013 15, 687-694DOI: (10.1016/j.jmoldx.2013.04.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Results for five DRT assays on DNA extracted from fixed and fresh melanoma cell lines, Sk-Mel-5 and Sk-Mel-28. The accuracy of DRT in fixed tissue is demonstrated by an intraclass correlation coefficient of 0.99 for agreement between DNA from fresh and fixed cells. The results of the predicted ratio, using the Predicting Integral Copy Number In Cancer application on array CGH data from these cell lines, are also plotted. The Sk-Mel-5 cell line is known to have a homologous deletion in the CDKN2A gene and there is, therefore, no predicted ratio from the array CGH data in this case. The Journal of Molecular Diagnostics 2013 15, 687-694DOI: (10.1016/j.jmoldx.2013.04.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Scatterplots of the DRT scores for the five assays, separated by class (tonsils, nevi, and melanoma). The Journal of Molecular Diagnostics 2013 15, 687-694DOI: (10.1016/j.jmoldx.2013.04.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions