ONHs were isolated from eyes harvested within six hours post mortem. The anterior portion of the eye and the vitreous were removed from the posterior eyecup. The ONH was excised using a 4mm trephine punch. A 2-3mm long portion of the adjacent optic nerve was included in the isolated material. Visible traces of the RPE and neural retina were removed from the excised ONH to avoid tissue contamination. Collected ONHs were stored at -80°C until RNA extraction.
Label
biotin
Label protocol
RNA was extracted using Qiagen RNeasy minipreps. RNA was treated with RNase free DNase and its integrity was evaluated using a 2100 BioAnalyzer microfluidics-based platform (Agilent, Palo Alto, CA). 50-100ng total RNA amplified using a T7 RNA polymerase based approach, reverse transcribed, dye labeled and hybridized to Affymetrix Human Exon 1.0 ST array according to the manufacturer’s protocol.
Hybridization protocol
Affymetrix protocol
Scan protocol
Affymetrix protocol
Description
C/D ratio 0.7, Alphagan user
Data processing
Data obtained were subjected to an initial QC analysis through Partek® Genomics Suite™ 6.6 using default program settings. Background adjustment, quantile normalization and mean probe set summarization to genes were performed using the Robust Multichip Average (RMA) algorithm. Normalized data were log2-transformed and filtered to remove non-expressed genes from the data sets. Expressed genes are defined as those represented by probe sets that display log-expression values above 7.0 in at least two samples. Gene expression values are reported in our analysis as the ratios for OHT and glaucoma samples relative to control samples. Differentially expressed gene sets in POAG and OHT versus control samples are identified using an analysis of variance (Djaoued, Robichaud et al.) paired sample t-test with a fold change cut off value of 1.25 and P-value < 0.05.
An Investigation of Global Gene Expression Patterns in Glaucoma and Ocular Hypertension Derived Optic Nerve Heads
Data table header descriptions
ID_REF
VALUE
Background adjustment, quantile normalization and mean probe set summarization to genes were performed using the Robust Multichip Average (RMA) algorithm, Partek® Genomics Suite™ 6.6.
