Quantitative Expression Profiling in Formalin-Fixed Paraffin-Embedded Samples by Affymetrix Microarrays Diana Abdueva, Michele Wing, Betty Schaub, Timothy.

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Quantitative Expression Profiling in Formalin-Fixed Paraffin-Embedded Samples by Affymetrix Microarrays Diana Abdueva, Michele Wing, Betty Schaub, Timothy Triche, Elai Davicioni The Journal of Molecular Diagnostics Volume 12, Issue 4, Pages 409-417 (July 2010) DOI: 10.2353/jmoldx.2010.090155 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Analysis of RNA from Wilm's tumor and paired kidney samples from FFPE and FF quadsets. A: Yields of RNA extracted from FF and FFPE samples archived for various block ages. B: Mean yields of amplified DNA from FFPE and FF generated using WT-Ovation FFPE kit. Yields are expressed as concentration of product obtained after two rounds of amplification using a combined random- and oligo-dT–primed protocol. Note for FFPE samples 50 ng was used as input, whereas for FF samples 10 ng input RNA was used as per manufacturer's recommendations. The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: Raw signal density, FFPE (blue) and FF (red). B: Scatterplot of log2 raw intensity of median FFPE normal samples (y axis) versus median FF normal samples (x axis). The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 The degradation plots, based on ordering probes within a probeset according to their 3′ position and then combining the signal from similarly located probes across the array. Each line corresponds to an array, red for FFPE and blue for FF, and the slope of its trend indicates potential RNA degradation and/or inefficient labeling. The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 A: Density function of probe contrast (i.e., median pairwise difference) in log2 FF and FFPE signal across normal (i.e., nontumorous) samples. Similar contrast function was observed in tumor samples. B: Density function of median probe contrast within probesets. C: Scatterplot of median versus maximum probe contrast within a probeset. The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 A: Density of median probe contrast within a probeset in kidney, lung, and colon tissues. B: Venn diagram of median within-probeset contrast larger than two in kidney, lung, and colon data sets. The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 A: Venn diagram, showing concordance of differential expression in gene expression of Wilm's tumor and normal adjacent kidney in FF and FFPE assays. B: Scatterplot of mean fold-difference between normal kidney and Wilm's tumor found significant in both FF and FFPE comparisons. C: Percent concordance in mean fold-difference between FF and FFPE as a function of mean fold-difference between Wilm's tumor and normal kidney in respective FF and FFPE assays. The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 7 A side-by-side view of P value threshold (x axis) in FFPE tumor/normal comparison versus concordance with FF differentially expressed gene list under nonstringent cut-offs (bottom) and fraction of genes reported (top). The Journal of Molecular Diagnostics 2010 12, 409-417DOI: (10.2353/jmoldx.2010.090155) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions