Heather N. Reich, Carol Landolt-Marticorena, Paul C

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Molecular Markers of Injury in Kidney Biopsy Specimens of Patients with Lupus Nephritis Heather N. Reich, Carol Landolt-Marticorena, Paul C. Boutros, Rohan John, Joan Wither, Paul R. Fortin, Stuart Yang, James W. Scholey, Andrew M. Herzenberg The Journal of Molecular Diagnostics Volume 13, Issue 2, Pages 143-151 (March 2011) DOI: 10.1016/j.jmoldx.2010.10.005 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Experimental workflow for mRNA expression analysis of FFPE 18-gauge renal biopsy specimens. Histological sections of archived FFPE biopsy specimens are cut and placed in a xylene-filled microtube for deparaffinization. After ethanol wash and protease digestion, RNA is isolated. RNA quantity and quality are measured and RNA is reverse transcribed to cDNA. The product is used either in a Taqman low-density array (TLDA) or standard Taqman real-time PCR. The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Effect of archival age on housekeeping gene expression in FFPE diabetes biopsy specimens. Expression of GAPDH and 18s housekeeping genes in 120 archived FFPE biopsy specimens were measured in triplicate using conventional real-time PCR. Mean amplification cycle thresholds (Ct) and SD are shown according to age of biopsy (A; archive time). For 8 years of archival time, the expression of GAPDH was relatively constant, and there was modest variability in expression levels. In contrast, there was a significant time-dependent decrease in 18s expression, and there was high variability in 18s expression in more recent biopsy specimens. B shows the time-dependent variability in 18s expression. The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Effect of qualitative biopsy grade on RNA quantity and purity. A: RNA concentration. B: RNA nanodrop spectrophotometry 260/280 ratios. C: RNA nanodrop spectrophotometry 260/230 ratios. Values are based on measurements in 60 biopsy samples (grade 1, n = 10; grade 2, n = 25; grade 3, n = 25). The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Storage-time effect on RNA concentration and purity in archived lupus FFPE renal biopsy specimens. A: RNA concentration was significantly higher in more recent biopsy specimens (P < 0.05). B: The 260/280 ratio did not differ according to biopsy vintage. C: The 260/230 ratio was superior in the most recent biopsy specimens (*P < 0.05) 2000 to 2002 (n = 8), 2003 to 2005 (n = 29), and 2006 to 2008 (n = 21). The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 TLDA housekeeping gene expression lupus biopsy specimens. A: Cycle threshold (Ct) of 16 housekeeping genes measured using the preformatted TLDA housekeeping gene card platform in eight samples in triplicate. A lower cycle threshold indicates higher mRNA abundance. B: Housekeeping gene expression stability using geNorm analysis; reduced gene expression variability compared to other housekeeping genes is reflected by lower score. Cyclophilin A (PPIA) shows the lowest relative variability (most stability). C: Expression of GAPDH compared with cyclophilin A in an additional 40 lupus samples (PPIA), measured using TLDA-based real-time PCR. There is a high correlation between GAPDH and cyclophilin A expression (r = 0.93, P < 0.05). The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Relationships between mRNA expression and biopsy chronicity scores. A: COL1A1 mRNA. B: MMP7 mRNA. C: EGF mRNA. D: Predicted versus actual chronicity. Analyses are based on mRNA expression measured in duplicate from 54 biopsy specimens. The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Representative histological sections and immunohistochemical staining. A and B: There is a direct correlation between COL1A1 protein and mRNA expression, and between COL1A1 protein expression and chronicity score. Biopsy specimens with low chronicity index (A, those with lesser degree of tubulo-interstitial fibrosis) showed low levels of COL1A1 protein expression, and biopsy specimens with a high chronicity index (B) showed high levels of COL1A1 expression. C and D: MMP 7 protein expression is not abundant in biopsy specimens of patients who have low MMP7 mRNA expression and low disease chronicity scores (C); however, there is abundant tubular staining for MMP7 protein in biopsy specimens with high mRNA expression and high chronicity indices (D). E and F: EGF protein expression is low in biopsy specimens with low EGF mRNA expression and high chronicity score (E), but is high in biopsy specimens with high mRNA expression and low chronicity score (F) (direct correlation with mRNA expression, inverse correlation with chronicity score). The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 8 Relationships between protein expression and chronicity index. The correlations between semiquantitative measurement of COL1A1 (A), MMP7 (B), and EGF (C) protein expression and biopsy chronicity scores are shown. Analyses are based on immunohistochemistry staining quantified in biopsy specimens (COL1A1, n = 19; MMP7, n = 19; and EGF, n = 7). The Journal of Molecular Diagnostics 2011 13, 143-151DOI: (10.1016/j.jmoldx.2010.10.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions