|
| Status |
Public on Mar 24, 2010 |
| Title |
Comparative transcriptional profiling of Limbal Epithelial Crypts with rest of the ocular surface epithelial regions. |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
|
| Summary |
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.
|
| |
|
| Overall design |
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.
|
| |
|
| Contributor(s) |
Kulkarni BB, Tighe PJ, Dua HS |
| Citation(s) |
20920242 |
| Submission date |
Nov 16, 2009 |
| Last update date |
Sep 28, 2015 |
| Contact name |
Patrick Jason Tighe |
| Organization name |
The University of Nottingham
|
| Department |
School of Molecular Medical Sciences
|
| Lab |
Immunology
|
| Street address |
University Hospital
|
| City |
Nottingham |
| State/province |
Nottinghamshire |
| ZIP/Postal code |
NG7 2UH |
| Country |
United Kingdom |
| |
|
| Platforms (1) |
|
| Samples (18)
|
| GSM471147 |
84390/CO398 Cornea+SP1/Biological cornea replicate 1 |
| GSM471148 |
84549/CO399 Cornea+SP1/Biological cornea replicate 2 |
| GSM471149 |
84544/CO418 Cornea+SP1/Biological cornea replicate 3 |
| GSM471150 |
84403/CO398 Limbus+SP1/Biological Limbus replicate 1 |
| GSM471151 |
84409/CO399 Limbus+SP1/Biological Limbus replicate 2 |
| GSM471152 |
84412/CO404 Limbus+SP1/Biological Limbus replicate 3 |
| GSM471153 |
84313/CO418 Limbus+SP2/Biological Limbus replicate 4 |
| GSM471154 |
84411/CO398 LEC+SP1/Biological LEC replicate 1 |
| GSM471155 |
84320/CO399 LEC+SP2/Biological LEC replicate 2 |
| GSM471156 |
84542/CO404 LEC+SP2/Biological LEC replicate 3 |
| GSM471157 |
84315/CO418 LEC+SP1/Biological LEC replicate 4 |
| GSM471158 |
84318/CO399 Conjunctiva+SP2/Biological conjunctival replicate 1 |
| GSM471159 |
84571/CO404 Conjunctiva+SP1/Biological Conjunctival replicate 2 |
| GSM471160 |
84550/CO418 Conjunctiva+SP2/Biological Conjunctival replicate 3 |
| GSM471161 |
84410/CO398 LEC Stroma+SP1/Biological LEC Stromal replicate 1 |
| GSM471162 |
84317/CO399 LEC Stroma+SP2/Biological LEC Stromal replicate 2 |
| GSM471163 |
84543/CO404 LEC Stroma+SP2/Biological LEC Stromal replicate 3 |
| GSM471164 |
84316/CO418 LEC Stroma+SP1/Biological LEC Stromal replicate 4 |
|
| Relations |
| BioProject |
PRJNA120639 |
Less...
More...