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Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis  Shuji Ogino,

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Presentation on theme: "Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis  Shuji Ogino,"— Presentation transcript:

1 Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis  Shuji Ogino, Takako Kawasaki, Mohan Brahmandam, Mami Cantor, Gregory J. Kirkner, Donna Spiegelman, G. Mike Makrigiorgos, Daniel J. Weisenberger, Peter W. Laird, Massimo Loda, Charles S. Fuchs  The Journal of Molecular Diagnostics  Volume 8, Issue 2, Pages (May 2006) DOI: /jmoldx Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Precision study for sodium bisulfite conversion and MethyLight. Bisulfite conversion was performed on seven different aliquots from each of the four cell lysate samples (cases 1 through 4), starting on 7 different days. Each sample was assessed for PMR at each locus by the MethyLight assay. MethyLight assays were repeated for each bisulfite-treated DNA sample on 5 different days, and PMR values were again determined. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 DNA mixing study to assess linearity of MethyLight. Human peripheral blood leukocyte DNA (Promega) was first treated with M. SssI to methylate all CpG sites (near complete methylation and no loss of DNA was assumed). Mixtures of SssI-treated DNA:untreated DNA were prepared in triplicate (each containing 1 μg of template DNA) as shown. One hundred percent SssI-treated DNA served as methylated reference DNA. Each DNA mixture was bisulfite-treated and assayed in duplicate by MethyLight. Linear regression analysis was performed to assess assay linearity. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 DNA mixing study and linear regression analysis to assess linearity of MethyLight. DNA mixing assays were performed as described in Materials and Methods and Figure 2. The 100% SssI-treated DNA sample served as methylated reference DNA to calculate PMR. Linear regression analysis to assess assay linearity was performed. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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