Hg u133a 2.0 arrays

For each case, the breast pathologist identified tumor areas for laser microdissection from H&E stained slides. Two to five serial sections (8 μm thick) were cut, mounted on glass PEN foil slides (Leica Microsystems, Wetzlar, Germany), stained using the LCM staining kit (Applied Biosystems, Foster City, CA) and laser microdissected on an ASLMD laser microdissection system (Leica Microsystems, Wetzlar, Germany). Slide preparation, staining and cutting were performed within a 15 min period to preserve RNA integrity. RNA was then isolated using the RNAqueous-Micro kit (Applied Biosystems, Foster City, CA) and treated with DNase I to remove any contaminating genomic DNA. RNA integrity was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), converted to biotin-labeled aRNA using two rounds of amplification with the MessageAmpII aRNA Amplification kit (Applied Biosystems, Foster City, CA), and the concentration and quality of the samples was measured with the NanoDrop 1000 (NanoDrop Products, Wilmington, DE) and 2100 Bioanalyzer. Hybridization with manufacturer provided hybridization controls, washing, staining and scanning of HG U133A 2.0 arrays (Affymetrix, Santa Clara, CA) were conducted according to manufacturer’s protocols [28 ].

Data were downloaded from GEO (GSE10240). Primary HBECs provided by the Tissue Core Laboratory at the University of Pittsburgh (Pittsburgh, Pennsylvania, USA) or purchased from Cambrex (Lonza) were grown to confluence in ALI, then stimulated apically and basolaterally with media control or IL-17 for 24 hours (3 replicates each) as previously described (43 (link)). Isolated RNA was profiled by Affymetrix HG U133A 2.0 Arrays.