RNA was isolated from alveolar macrophages using the MirVana (Applied Biosystems (ABI), Austin, TX) reagents according to the manufacturer's instructions.
Label
biotin
Label protocol
Microarray hybridizations were performed at the University of Iowa DNA Facility. Briefly, 25 nanograms total RNA was converted to SPIA amplified cDNA using the WT-Ovation Pico RNA Amplification System, v1 (NuGEN Technologies, San Carlos, CA, Cat. #3300) according to the manufacturer’s recommended protocol. The amplified SPIA cDNA product was purified through a QIAGEN QIAquick PCR Purification column (QIAGEN Cat #28104) according to modifications from NuGEN. Four ug of SPIA amplified DNA were 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 were 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.
Hybridization protocol
Biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer (Affymetrix, Inc., Santa Clara, CA), placed onto Affymetrix Human Exon 1.0 ST arrays (Part No. 900650), and incubated at 45º C for 18 h with 60 rpm rotation in an Affymetrix Model 640 GeneChip Hybridization Oven. Following hybridization, the arrays were washed, stained with streptavidin-phycoerythrin (Molecular Probes, Inc., Eugene, OR), and signal amplified with anti-streptavidin antibody (Vector Laboratories, Inc., Burlingame, CA) using the Affymetrix Model 450 Fluidics Station.
Scan protocol
Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade, and data were collected using the GeneChip Operating Software (GCOS) v1.4.
Data processing
CEL files were imported into PartekGS version 6.5 and normalized using the standard RMA workflow. The gene values were obtained using mean value summarization.
Genome build: hg18 Exons used in analysis: core Probe group file: HuEx-1_0-st-v2.r2.pgf Meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps
