The A549 cells (ATCC CCL 165), a human lung carcinoma cell line, were grown in RPMI medium 1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 24 mM HEPES and 2 mM L-glutamine and maintained at 37 degrees C in 5% CO2. Cells that were passaged 86-99 times were used for all experiments. Prior to each experiment, the cells were washed 2 times in Hank’s Balanced Salt Solution and incubated for 12-14 hrs in serum-free RPMI medium 1640 supplemented with 24 mM HEPES and 2 mM L-glutamine. The media was replaced again with fresh serum-free media just prior to addition of bacteria. The bacteria were grown in Luria-Bertani broth until mid-log phase (OD600 ~2.0), gently washed twice in PBS and then added to the flask containing A549 cells at a multiplicity of infection of 50:1 bacterial to A549 cells. Total RNA was isolated as previously described. PolyA enrichment was accomplished by oligo(dT)-cellulose selection using the Poly(A)Pure Kit (Ambion). PolyA enriched RNA was used to synthesize double stranded cDNA according to the Affymetrix supplied protocol (Affymetrix, Inc.). The first strand synthesis reaction was accomplished using 2 mcg RNA, SuperScript II Reverse Transcriptase (Invitrogen) and T7-(dT)24 primer (Genset Corp.) A second strand synthesis reaction followed using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H (Invitrogen). The double stranded cDNA was phenol extracted and used as template in an in vitro T7 transcription reaction using the MEGAscript T7 high yield transcription kit (Ambion) and biotinylated nucleotides Biotin-11-CTP, Biotin-16-UTP (NEN, Perkin-Elmer, Boston, MA). A 20 mcg cRNA aliquot was fragmented in 1X fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) at 94 degrees C for 35 min. To check the quality of each sample, the cDNA, full length and fragmented cRNA were analyzed by electrophoresis on a 1% Agarose-TAE gel. 15 mcg of the fragmented cRNA were hybridized to an Affymetrix human GeneChip probe array U95Av2 for 16 hours at 45 degrees C. The probe arrays were washed, stained and scanned in an Affymetrix fluidics station and scanner following the manufacturer’s protocols. Raw image analysis was performed using the Affymetrix Microarray Suite v5.0 software. Keywords = Pseudomonas aeruginosa Keywords = A549 human lung carcinoma cell line Keywords = cystic fibrosis
