GSE113008 mRNA sequencing of Aniridia limbal epithelial cells.

Contributors : Karl Nordstr öm ; Jasmin KirchSeries Type : Expression profiling by high throughput sequencingOrganism :PAX6-related Aniridia is a sight-threatening disease due to progression of secondary glaucoma and aniridia associated keratopathy (AAK). Changes or loss of limbal epithelial progenitors causes the epithelial surfaces defects. We analyzed how PAX6 contribute to this with a two-step approach. 1) mRNA Sequencing of limbal epithelial cells isolated from controls and aniridia patients. 2) confirming the bioinformatical and literature-based result list on a siRNA based primary aniridia cell model (PAX6- knockdown). With this approach genes which are directly influenced by PAX6 should be distinguishable from genes secondary affected by AAK disease state. Therefore epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium. Normal cells were obtained from limbus region of cadaveric control patients. For the siRNA based anridia cell model cells were transfected with lipofectamine and 5 nM siRNA against PAX6 or scrambled control. All cells were lysed to obtain DNA, RNA and protein. Reduction of PAX6 protein was controlled by Western Blot. Aniridia and Control Poly-A enriched RNA-librarys were subjected to Next Generation Sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparing to NCBI GEO Datasets, annotation with D...
Source: GEO: Gene Expression Omnibus - Category: Genetics & Stem Cells Tags: Expression profiling by high throughput sequencing Source Type: research