normalization data transformation protocol
Ten Affymetrix GeneChip Human Exon 1.0 ST (HuEx 1.0) microarrays were simultaneously normalized using the Affymetrix Power Tools (APT) probeset summarization tool. Normalization was done at both the probeset and metaprobeset level using the PLIER-sketch method with GC-background correction. HuEx-1_0-st-v2.r2.dt1.hg18.core.ps HuEx-1_0-st-v2.r2.dt1.hg18.core.mps ID_REF = VALUE = PLIER-GCBG corrected estimates from Affymetrix Power Tools
array scanning protocol
Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade and data collected using the GeneChip operating software (GCOS) v1.4.
The resulting biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer (Affymetrix, Inc., Santa Clara, CA), placed onto Affymetrix Human Exon 1.0 ST Arrays, and incubated at 45º C for 18 h with 60 rpm rotation in an Affymetrix Model 640 Genechip Hybridization Oven.
Four ug of SPIA amplified DNA was then used to generate ST-cDNA using the WT-Ovation Exon Module v1 (NuGEN Technologies, Cat #2000) and again cleaned up with the Qiagen column as above. Five micrograms of this product was fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module, v2 (NuGEN Technologies, Cat. #4200) per the manufacturer’s recommended protocol.
nucleic acid extraction protocol
Total RNA was extracted using Trizol Plus RNA Purification kits (Invitrogen) as previously described by Wang W-H, et al.
sample treatment protocol
Human donor eyes (20 normal donors) were obtained from the Lions Eye Institute for Transplant and Research (Tampa, FL) and stored in RNALater (Invitrogen) at 4 °C for up to 30 d. Tissue was dissected under a microscope into cornea, iris, ciliary body, trabecular meshwork, lens, retina, choroid/RPE, sclera, optic nerve, and optic nerve head, and stored at -80 °C.