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Ricardo F Frausto, Derek Le, Anthony J Aldave; High-throughput gene expression analysis of the human cornea. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4919.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the transcriptome profile for each of the three primary cell types comprising the human cornea using next-generation sequencing technology (NGS).
Total RNA was isolated from ex vivo corneal epithelium (HCEpC), stromal fibroblasts (HCFC) and endothelium (HCEnC), obtained from eye bank donor corneas. Purified RNA samples were submitted to the UCLA Microarray Core and underwent RNA-sequencing. NGS data were analyzed using Partek Genomics Suite and Partek Flow software. Briefly, reads were aligned to the hg38 genome build, quantified using the RPKM-method and annotated using Ensemble 77. Multidimensional data analysis was performed using principal component analysis (PCA) and hierarchical clustering (HC). Differential gene expression was determined using a false discovery rate (FDR)-adjusted p-value of 0.05 and fold-change of 5. Gene ontology (GO) enrichment was used to identify significantly enriched (p-value < 0.05) GO terms associated with gene-sets specific to each cell type.
Multidimensional data analysis using PCA and HC demonstrated three distinct populations of samples that grouped together based on cell type. There were 337, 164 and 139 uniquely expressed genes in epithelium, stromal fibroblasts and endothelium, respectively. Subsequently, GO enrichment analysis resulted in the identification of unique functional groups associated with each cell type. Of the 119 GO terms that were significantly enriched in HCEpC, 100 were unique to epithelium, including desmosome, retinoic acid metabolism process and epithelial cell differentiation. HCFC were significantly enriched for 128 GO terms, of which 111 were unique to this cell type, including metallopeptidase activity, collagen catabolic process and positive chemotaxis. HCEnC were significantly enriched for 65 GO terms, with 45 being unique to endothelium, such as carbonate dehydratase activity, beta-catenin binding and establishment of cell polarity.
This is the first comprehensive study investigating the transcriptome of ex vivo HCEpC, HCFC and HCEnC, the three main cell types of the human cornea. Transcriptome analysis revealed that on average 213 genes were uniquely expressed in each corneal cell type. These results, coupled with our functional enrichment data, suggest that a relatively small number of genes confer distinct functional properties that define each cell type.
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