RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. RNA was extracted from three additional and independent groups of 12-month-old MUT and WT mice, respectively, and from human samples using TRIzol and RNeasy spin column (Qiagen) purification. Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was performed through the Affymetrix GeneChip Command Console version 2.0 (AGCC v2.0) software. Affymetrix Fluidics Station 450 was used to wash and stain the arrays, removing the nonhybridized target and incubating with a streptavidin–phycoerythrin conjugate to stain the biotinylated cDNA. Five micrograms of ST-cDNA were subsequently enzymatically fragmented, biotin labeled using the Encore Biotin module (NuGen Technologies), and hybridized onto Mouse Exon 1.0 ST arrays (Affymetrix) for 18 hours at 45☌ with constant rotation at 60 rpm. Sense transcript cDNA (ST-cDNA) was created from 3 μg of purified cDNA using the WT-Ovation Exon module (NuGen Technologies). The Ovation Pico WTA system (NuGEN Technologies, San Carlos, CA) was used to generate SPIA-amplified cDNA from 10 ng of RNA. Gene array analysis in endothelial RNA samples was performed on Mouse Exon 1.0 ST microarrays (Affymetrix, Santa Clara, CA). In particular endothelial COX2 up-regulation warrants further investigation of its role in FECD. The present study provides the first endothelial whole genome expression analysis in an animal model of FECD and represents a useful resource for future studies of the disease. In human FECD samples, real-time PCR demonstrated a statistically significant increase in COX2 mRNA ( P < 0.0001) and JUN mRNA ( P = 0.002) and tissue microarray analysis showed increased endothelial COX2 ( P = 0.02) and JUN protein ( P = 0.04). Real-time PCR assessment confirmed increased Cox2 ( P = 0.001) and Jun mRNA ( P = 0.03) levels in Col8a2 Q455K/Q455K mutant compared to wild-type mice. Microarray analysis demonstrated endothelial expression of 24,538 genes (162 up-regulated and 172 down-regulated targets) and identified affected gene ontology terms including Response to Stress, Protein Metabolic Process, Protein Folding, Regulation of Apoptosis, and Transporter Activity. Endothelial COX2 and JUN expression was further studied in human late-onset FECD compared to normal samples. Result validation was performed by real-time PCR. An endothelial whole genome expression profile was generated by microarray-based analysis. To investigate the endothelial gene expression profile in a Col8a2 Q455K mutant knock-in mouse model of early-onset Fuchs' endothelial corneal dystrophy (FECD) and identify potential targets that can be correlated to human late-onset FECD.ĭiseased or normal endothelial phenotypes were verified in 12-month-old homozygous Col8a2 Q455K/Q455K mutant and wild-type mice by clinical confocal microscopy.
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