Precision Profiling and Components of Variability Analysis for Affymetrix Microarray Assays Run in a Clinical Context Thomas M. Daly, Carmen M. Dumaual,

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Precision Profiling and Components of Variability Analysis for Affymetrix Microarray Assays Run in a Clinical Context Thomas M. Daly, Carmen M. Dumaual, Crystal A. Dotson, Mark W. Farmen, Sunil K. Kadam, Richard D. Hockett The Journal of Molecular Diagnostics Volume 7, Issue 3, Pages 404-412 (August 2005) DOI: 10.1016/S1525-1578(10)60570-3 Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Experimental designs for studies performed in this article. A: U95Av2 variability assessment study. Diagram represents a single run of eight chips performed by two analysts. *Fluidic stations were varied between analysts on alternate runs. B: U133A reagent lot study. Diagram represents a single run of four chips performed by one analyst. *Hybridization mix lots were reversed on alternate runs to incorporate all possible reagent lot combinations. C: U133A tumor matrix study. Diagram represents a single run of four chips performed by one analyst. Reagent lots were held constant for all runs in this study. The Journal of Molecular Diagnostics 2005 7, 404-412DOI: (10.1016/S1525-1578(10)60570-3) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: Concordance of P+M calls across the 55 chips analyzed. The majority of probesets were consistently called either P+M or A on all chips. B: Coefficient of variation of signal intensity was calculated for each probeset, and plotted against the mean intensity for all probesets. C: Expanded view of CV versus intensity for present probesets only. D: Probeset CV as a function of median P value. Dotted line indicates P = 0.06. The Journal of Molecular Diagnostics 2005 7, 404-412DOI: (10.1016/S1525-1578(10)60570-3) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Components of variability analysis. Probesets are divided into present (median P value <0.06) or absent (median P value >0.06) for each study. Mean with SD error bars are plotted. A: U95Av2 variability assessment study. B: U133A reagent lot study. The Journal of Molecular Diagnostics 2005 7, 404-412DOI: (10.1016/S1525-1578(10)60570-3) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Probeset variability of U133A Affymetrix chips. A: Coefficient of variation versus signal intensity for both present and absent probesets. B: Comparison of CVs between U95Av2 and U133A chips for probesets called present. The Journal of Molecular Diagnostics 2005 7, 404-412DOI: (10.1016/S1525-1578(10)60570-3) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Comparison of U133A probeset CVs from two different materials. A: The CVs for the 7434 probesets present in both the cell line and tumor RNA samples are compared. Quadrants are divided at 40% CV for each sample, percentages indicate the number of probesets in that quadrant. B: Relative signal intensities of probesets that performed differently in the two samples (A: top left and bottom right). Diagonal line represents equal intensity. Probesets tended to have lower signal intensities in the sample with the higher CV. The Journal of Molecular Diagnostics 2005 7, 404-412DOI: (10.1016/S1525-1578(10)60570-3) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions