Four explants from each lamina cribrosa were dissected and placed into 25-cm2 Primaria tissue culture flasks and maintained in DMEM-F12 supplemented with 10% FBS and antibiotics.Cells were kept in a 37oC, 5% CO2 incubator. After 2-4 weeks, primary cultures were purified by using modified immunopanning procedure described by Mi and Barres (1999). Purified cells were expanded after characterization by immunostaining for astrocyte markers GFAP and NCAM as described (Yang and Hernandez, 2003). Second passage cell cultures were stored in RPMI 1640 with 10% DMSO in liquid nitrogen until use.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Qiagen RNeasy mini kits (Qiagen, Valencia, CA). RNA was then purified and quantified by measuring absorbance at 260 nm. Quality and intactness of the RNA was assessed by capillary electrophoresis analysis using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).
Label
biotin
Label protocol
cRNA was synthesized from 2-5 µg purified RNA by using Superscript Choice system (Gibco BRL Life Technologies, Gaithersburg, MD ) and T7-(dT)24 primer (GENSET, La Jolla, CA). Using Bioassay High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), in vitro transcription was carried out by using the cleaned double-stranded cDNA as a template in the presence of biotinylated UTP and CTP.
Hybridization protocol
Purified biotin-labeled cRNA was fragmented before the hybridization. Hybridization of the labeled cRNA to Human Genome U133A and U133A 2.0 chips (Affymetrix, Santa Clara, CA) was carried out by using Genechip Instrument System (Affymetrix) at the Genechip Core Facility of Washington University (Saint Louis, MO).
Scan protocol
The arrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR) followed by scanning on an Agilent GeneArray Scanner G2500A (Agilent Technologies, Palo Alto, CA), and then scanned by an Affymetrix Genearray Scanner.
Description
LC76c Caucasian American
Data processing
The Hu133av2 and Hu133a data were first separately normalized by using RMA method. Then the data were combined based on the common probeset Ids (Hu133a's Probeset Ids) and followed by quantile normalization to reduce the batch effects.
