In Vitro Transcription Amplification and Labeling Methods Contribute to the Variability of Gene Expression Profiling with DNA Microarrays Changqing Ma,

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In Vitro Transcription Amplification and Labeling Methods Contribute to the Variability of Gene Expression Profiling with DNA Microarrays Changqing Ma, Maureen Lyons-Weiler, Wenjing Liang, William LaFramboise, John R. Gilbertson, Michael J. Becich, Federico A. Monzon The Journal of Molecular Diagnostics Volume 8, Issue 2, Pages 183-192 (May 2006) DOI: 10.2353/jmoldx.2006.050077 Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Concordance on present/absent transcript calls among the four methods studied. Total number of transcripts in the U95v2 array is 12,592. Complete agreement among methods is represented by a white background and further divided into present and absent calls. Disagreement among methods is shaded. The Journal of Molecular Diagnostics 2006 8, 183-192DOI: (10.2353/jmoldx.2006.050077) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Variability in gene expression data. a: Intra- and interassay CV for all present transcripts. The solid line on each box represents the median CV and the dashed line represents the mean CV. b: Example of two transcripts with high-intensity values in hybridization results showing no change across methods. c: Example of one transcript with low-intensity values showing difference between Affy versus Affy4h. d: Example of low expressor affected by the Enzo method in the Enzo versus Affy4h. The Journal of Molecular Diagnostics 2006 8, 183-192DOI: (10.2353/jmoldx.2006.050077) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Role of double-nucleotide labeling in the variability of gene expression data. Enzo- and Affy4h-affected transcripts (totally 5085) were divided into groups based on the guanine to adenine (G/A) ratio in the target sequence, reflecting cytosine to uracil incorporation into the cRNA transcript. In each group, the number of affected transcripts by each method and the corresponding percentages of total number of affected transcripts in this group were obtained. The percentage of affected transcripts of each method in each group is plotted. The Journal of Molecular Diagnostics 2006 8, 183-192DOI: (10.2353/jmoldx.2006.050077) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Role of transcript length in the variability of gene expression data. Affected transcripts from SAM analysis of the Affy versus Affy4h comparison were grouped based on their transcript lengths. The proportion of affected transcripts for each method in each transcript length interval is plotted. Please note only the transcripts shorter than 1.5 kb were of interest to this analysis. The Journal of Molecular Diagnostics 2006 8, 183-192DOI: (10.2353/jmoldx.2006.050077) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions