Whole Genome Amplification for Array Comparative Genomic Hybridization Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Histological Sections

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Whole Genome Amplification for Array Comparative Genomic Hybridization Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Histological Sections Jian Huang, Jesse Pang, Takuya Watanabe, Ho-Keung Ng, Hiroko Ohgaki The Journal of Molecular Diagnostics Volume 11, Issue 2, Pages 109-116 (March 2009) DOI: 10.2353/jmoldx.2009.080143 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Agarose gel electrophoresis of WGA-DNA (0.9% agarose gel). WGA was performed with Phi29 (reaction time of 1.5 hours) and with 10 ng of template DNA for lambda DNA (P) and 300 ng for DNA extracted from tumors 1 and 2 on paraffin sections. For DNA from tumors 1 and 2, a ligation step was added before WGA. Note that WGA-DNA samples from tumor 1 and tumor 2 show similar amounts and similar molecular weights of DNA (0.5 ∼ 20 kb) with WGA-lambda DNA. M, 1-kb DNA ladder marker; N, negative control (H2O), showing that the negative control does not generate DNA product. The Journal of Molecular Diagnostics 2009 11, 109-116DOI: (10.2353/jmoldx.2009.080143) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Top: Mean yield of Phi29-based WGA-amplified DNA. Note that the reaction time but not the amount of template DNA affects the yield. In samples without ligation, there was no WGA product when the reaction time with Phi29 was less than 1 hour, irrespective of the amount of template DNA. Bottom: Amplification bias in WGA products calculated from the data on copy number of six markers (D10S536 and D10S1683 at 10q, D1S2736 and D1S468 at 1p, and D19S408 and D19S596 at 19q) in WGA-amplified DNA (see Materials and Methods). Samples with a ligation step showed lower amplification bias in WGA products compared with samples without ligation. Error bars represent SD. Note that a higher amount of template DNA and shorter reaction time with Phi29 generate lower amplification bias. *No production of WGA-DNA. The Journal of Molecular Diagnostics 2009 11, 109-116DOI: (10.2353/jmoldx.2009.080143) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Array-CGH results using DNA before and after WGA. The genome profiles of selected chromosomes 1, 12, 13, and 18 of tumor 2 show similar gain and loss patterns in samples before and after WGA, despite the fact that samples after WGA show high noise. The Journal of Molecular Diagnostics 2009 11, 109-116DOI: (10.2353/jmoldx.2009.080143) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Array-CGH analysis in WGA-DNA reproduced results on EGFR amplification at 7p12.1 in tumor 1. The Journal of Molecular Diagnostics 2009 11, 109-116DOI: (10.2353/jmoldx.2009.080143) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions