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Comparative gene expression in the limbus of the vervet monkey

ABSTRACT: The limbus is a unique anatomic structure that is highly innervated and vascularized yet sets the boundary for the avascularized cornea. The corneal epithelial stem cells are believed to reside at the limbus. The intrinsic and external molecular signals that modulate the differentiation and proliferation of the limbal stems cells are still largely unidentified because of a lack of known specific markers. In this study, we used microarray technology to identify the unique gene expression profile in the limbus by comparing directly to that of its immediate adjacent structures, the cornea and conjunctiva, in the vervet monkey (Chlorocebus aethiops sabaeus). Many new genes were found to be preferentially expressed in the limbus, and two new biological pathways, melanin metabolism and apoptosis, were among other previously known processes identified in the limbus. These findings may shed light on the molecular components of limbal stem cells and their niche. Keywords: transcription profiling, gene expression

ORGANISM(S): Homo sapiens Chlorocebus sabaeus

PROVIDER: GSE10683 | GEO | 2008/03/05

SECONDARY ACCESSION(S): PRJNA107671

REPOSITORIES: GEO

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Project description:Recently we had discovered a solid cord like structure at the limbus of human eyes, termed as the Limbal Epithelial Crypt (LEC). It arises from the undersurface of the interpalisade rete ridges and extends towards the conjunctiva over the conjunctival stroma. Anatomical and immunohistochemical studies have shown it to be potentially a Stem Cell Niche. To confirm this hypothesis we conducted comparative gene expression profile of LEC with pathway and Geneontology studies in comparison with other ocular surface epithelial regions such as cornea, limbus, LEC stroma and conjunctiva. Frozen tissue blocks of corneoscleral buttons dissected from cadaver eyes were cryosectioned. These tissue sections from different ocular surface regions were laser microdissected. Extracted RNA was amplified & hybridized to 30,000k Human spotted cDNA microarray chips. Raw data obtained with Genepix Pro6 software was filtered, normalized & analysed on BASE & Jexpresspro software. Unpaired T-Test, Significance Analysis of Microarrays were performed on the data. Database for Annotation, Visualisation, and Integrated Discovery (DAVID) (http://www.DAVID.niaid.nih.gov) and Ingenuity Pathway Analysis (IPA) was used to determine the enriched GO terms and pathways in the differentially expressed genes. Quantitative gene expression analysis (qPCR) and immunohistochemistry was performed on the genes of interest. Statistical analysis for real time PCR was performed on SPSS16 to determine the normalised expression of gene of interest on ocular surface regions. Samples were prepared from five human ocular surface epithelial regions such as Cornea, Limbus, LEC, LEC Stroma, Conjunctiva. There were four replicates in each groups except Cornea and Conjunctiva with 3 each. The Standard Probe (SP) was prepared from mixing equal amount of Corneal and conjunctival epithelial RNA followed by ethanol precipitation. Samples were labelled with Cy5 dye and Standard Probe with Cy3 Dye. These were mixed in equal amount to prepare hybrid probes which were then hybridised to microarray slide.

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Comparative gene expression in the limbus of the vervet monkey

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