BioProject

Gene expression in human optic nerve head (ONH) astrocytes exposed to either 60 mm Hg hydrostatic pressure (HP) or control ambient pressure (CP) was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. More...

Gene expression in human optic nerve head (ONH) astrocytes exposed to either 60 mm Hg hydrostatic pressure (HP) or control ambient pressure (CP) was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. Primary ONH astrocytes from two male Caucasian donors (passage 4) were grown to 75% confluence and were exposed for 6, 24 or 48 h to control ambient pressure (CP6, CP24, CP48) or hydrostatic pressure (HP6, HP24, HP48), or harvested at the beginning of the pressure experiment (CP0). Total RNA was extracted using Qiagen RNeasy columns and converted to biotin-labeled cRNA by standard Affymetrix protocols available at web site http://pathology.wustl.edu/~mgacore/genechip.htm#Preparing. Hybridization of the labeled cRNA to Human Genome U95Av2 chips (Affymetrix) was carried out by using Genechip Instrument System (Affymetrix) at Genechip Core Facility of Washington University. A total of 44 chips were generated and distributed as follows: seven for control pressure (CP) at time 0, four CP at 6 h, seven for hydrostatic pressure (HP) at 6 h, eight for CP at 24 h, eight for HP at 24 h, five for CP at 48 h and five for HP at 48h P. The arrays were washed and stained with streptavidin-phycoerythrin followed by scanning on an Agilent GeneArray Scanner G2500A (Agilent Technologies, Palo Alto, CA), and then scanned by an Affymetrix GeneArray Scanner. Data was analyzed by Affymetrix Microarray Suite (version 5.0), linear regression analysis, and GeneSpring expression Analysis Software (version 6.0, Silicon Genetics). For analysis, the samples were scaled to the same target intensity (1500) to allow comparison of multiple samples, and raw data were normalized to median of each gene across all chips for fold change analysis using GeneSpring. Keywords: time-course Less...