Total RNA was extracted and purified from the clinical lung specimens using the SV96 Total RNA Isolation System (Promega) according to a custom automated protocol. The extracted RNA was quantified using RiboGreen RNA Quantitation Reagent (Invitrogen) and its quality was assessed using Agilent RNA 6000 Pico Kit (Agilent, Santa Clara, CA) in an Agilent 2100 Bioanalyzer (Agilent).
Label
biotin
Label protocol
Samples were amplified and labeled using the Ovation WB protocol (NuGEN Technologies, San Carlos, CA), according to the manufacturer’s instructions. In brief, 50 ng of total RNA was amplified using the Ribo-SPIA technology (NuGEN Technologies). After quantification, 4.4 ug cDNA was fragmented and labeled with biotin using the FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies).
Hybridization protocol
The amplified cDNAs were hybridized to Affymetrix gene expression chips (Human Rosetta Custom Affymetrix 2.0, Affymetrix, Santa Clara, CA) using standard Affymetrix protocols.
Scan protocol
Arrays were scanned using a GeneChip Scanner 3000 7G. The images were analyzed using the Affymetrix GeneChip Operating Software (GCOS).
Data processing
Expression values were extracted using the Robust Multichip Average method (Irizarry RA et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003;4:249-64) as implemented in the Affymetrix Power Tools software (available at: http://www.affymetrix.com/partners_programs/programs/developer/tools/powertools.affx).
