Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of human post-mortem conjunctivae and corneal epithelia

ABSTRACT: Whole human conjunctivae and human corneas were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and processed between 48 - 72 hr of death. No donor details apart from age, sex and cause of death were released. Criteria for inclusion were donor ages between 25 - 65 years old and overt physical integrity of the epithelium over the central cornea as revealed by a quick stain with 0.1 % Trypan blue. For each replicate experiment (Conjunctiva 1 and Cornea 1; Conjunctiva 2 and Cornea 2;Conjunctiva 3 and Cornea 3), RNA from two different corneal or conjunctival specimens was pooled and converted into cDNA. Appropriately fragmented biotin-labeled cRNA was hybridized to the HG-U133A GeneChips® (Affymetrix) oligonucleotide microarray following the manufacture protocol. Three separate experiments each including one conjunctival and one corneal cRNA pool were performed (Conjunctiva 1 and Cornea 1; Conjunctiva 2 and Cornea 2;Conjunctiva 3 and Cornea 3).

ORGANISM(S): Homo sapiens

SUBMITTER: Jose Mario Wolosin

PROVIDER: E-GEOD-5543 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

Turner Helen C HC Budak Murat T MT Akinci M A Murat MA Wolosin J Mario JM

Investigative ophthalmology & visual science 20070501 5

<h4>Purpose</h4>To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.<h4>Methods</h4>cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with th ...[more]

PMID: 17460260

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Project description:PURPOSE. To determine global mRNA expression levels in the corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. METHODS. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing > 22,000 transcripts. The resulting signal intensities and transcript present/absent calls by the primary microarray analyzing software were used in conjunction with a statistical method to identify transcripts that are preferentially or exclusively (transcripts absent in all three replicates in the other tissue) and significantly (expression >1% of the ß-actin level) expressed in one of the two tissues. EASE was used to identify biological systems comparatively over-represented in either epithelium. Immuno-, and cyto-histochemistry was performed to validate or expand on selected the results of interest. RESULTS. Our analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding conjunctival epithelium values were 592 and 211. The over-represented biological processes in the cornea were related to cell adhesion and oxi-redox equilibria and cytoprotection activities. In the conjunctiva the biological processes that were most prominent were related to innate immunity and to melanogenesis. Immunohistochemistry for antigen presenting cells and melanocytes were consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not previously been identified or not known to be highly expressed in these two epithelia, including, testican-1, ECM1, a forming isoform, CRTAC1 and NQO1 in the cornea and sPLA2-IIA, multiple MCH class II proteins, IGFBP3, and Na-Pi cotransporter type IIb in the conjunctiva. CONCLUSION. Comparative gene expression profiling led to the identification of many biological processes and previously unknown genes potentially important for the function of corneal and conjunctival epithelia. Keywords: Cell type comparison

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.

Dataset Information

Transcription profiling of human post-mortem conjunctivae and corneal epithelia

Publications

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

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