Project name: Rattus norvegicus

Description: In this work, we tested feasibility of high content screening of acutely isolated RGCs to generate systems biology knowledge of intrinsic cellular pathways associated with the onset of glaucomatous RGC loss in the retina. We performed differential profiling of these neurons using two complementary techniques: proteomics and microarray-based transcriptomics. The analysis of these RGC-specific proteomic and transcriptomic data using pathway informatics databases and biological network tools have revealed complex metabolic and regulatory changes in the COH-challenged neurons. We used the iTRAQ reagents that allow for the identification and quantitation of up to four different samples simultaneously, to assess the COH-induced changes in protein content of adult rat RGCs. Similar principle is utilized in the two-color Agilent arrays that we used for our transcriptomic analysis of the same RGC samples.Overall design: We performed high-content analyses of molecular changes in retinal ganglion cells associated with chronic ocular hypertension (COH). Gene expression profiling was performed using two-color microarrays. To assess differences in the gene expression levels in COH-challenged vs. normotensive RGCs, the “glaucomatous RGC/ reference RNA” ratios were divided by the ratios of “normotensive RGCs /reference RNA” obtained from the same group of experimental animals. Equivalent amounts of reference and experimental aRNAs were hybridized with the Agilent Rat Oligo Microarrays (Agilent, USA) according to the manufacturer’s instructions. Each microarray was scanned and normalized for signal intensities across the whole array and locally, using the Lowess normalization. We analyzed only those genes that passed Agilent quality control criteria. To reveal the genes with significant changes in glaucoma, we first calculated ratios of expression levels in experimental vs. fellow control eyes for each of four independent groups, and then selected genes with consistent changes using ANOVA and the cut-off p-value <0.05. Within each group, experimental and control RNA was extracted from RGCs purified from eye pairs originating from the same animal. Within each pair, as well as within each pooled group, total RNA abundances were, therefore, normalized for individual differences, so as were the resultant gene expression ratios. A fold change difference ≥1.5 was used to determine differentially expressed genes.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Release date: 2009-08-20

Last updated: 2009-03-22