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Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling  Anna E. Szafranska, Timothy S. Davison,

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Presentation on theme: "Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling  Anna E. Szafranska, Timothy S. Davison,"— Presentation transcript:

1 Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling  Anna E. Szafranska, Timothy S. Davison, Jaclyn Shingara, Martina Doleshal, Judith A. Riggenbach, Carl D. Morrison, Scott Jewell, Emmanuel Labourier  The Journal of Molecular Diagnostics  Volume 10, Issue 5, Pages (September 2008) DOI: /jmoldx Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 miRNA expression profiling in four mouse tissues. A: Unsupervised hierarchical clustering of miRNA expression in paired frozen and fixed mouse brain, stomach (Stom), small intestine (SmInt), and kidney (Kidn) using a two-color array platform. Expression levels are color-coded from green to red relative to the common reference hybridization sample used for each array, with red indicating enrichment in a given sample. Gray squares represent flagged miRNAs probes with low array signal below threshold (no data). B and C: Correlation of miRNA expression levels for paired fixed versus frozen mouse kidney, brain, small intestine, and stomach. Correlations were calculated using mean normalized expression levels in fixed and frozen samples (n = 4 donors per tissue). The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Correlation between fixed and frozen miRNA expression profiles in myometrium according to tissue age. A–C: Mean miRNA expression in 1-, 7-, and 11-year-old FFPE myometrium samples relative to frozen samples. The correlation value in brackets was calculated following removal of miR-494 and miR-513. D: Mean expression level of miR-494 and miR-513 in indicated fixed samples, relative to expression levels in frozen samples. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Variation of microarray platform performance according to FFPE tissue block age. A: Mean percentage of miRNAs detected above threshold for frozen (Myo_(1yr), n = 2), fixed 1-year-old (Myo_(1yr), n = 2), fixed 7-year-old (Myo_(7yr), n = 3), and fixed 11-year-old (Myo_(11yr), n = 4) myometrium tissues. The minimum threshold was defined as the 5% symmetric trimmed mean plus two standard deviations of the foreground minus background median intensities across all empty spots on individual arrays. B: Average foreground signals (excluding control spots) and background signals (including all spots) for microarrays described in A. C: Mean percentage of miRNAs detected above threshold in fixed myometrium samples according to expression levels in frozen samples. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5 Figure 4 Differential miRNA expression for paired fixed versus frozen myometrium (n = 6 samples) and B-cell lymphoma (n = 6 samples). A: Principal component analysis of miRNA expression in fixed and paired frozen myometrium and B-cell lymphoma. B: Correlation between mean differential miRNA expression level (Δh) in fixed myometrium and B-cell lymphoma as compared to corresponding frozen tissue set. The indicated correlation coefficient includes probes that were detected in any of the eight analyzed samples. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

6 Figure 5 Comparison between microarray and qRT-PCR platforms. A: Example of miRNAs differentially expressed in three independent liver NAT and spleen technical replicates as determined by microarray and qRT-PCR. For each RNA sample, duplicate reverse transcription reactions followed by duplicate PCR from each reverse transcription reaction was performed. B: Correlation between the microarray and qRT-PCR platforms for the mean differential expression of 14 miRNAs in liver NAT and spleen replicates. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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