Biomaterial manipulations: P. aeruginosa was grown in Brain-Heart-Infusion (BHI) medium at 37°C with shaking with or without the addition of 2 µg/ml AZM (Pfizer, Germany).
RNA extraction, cDNA synthesis and labeling: Total RNA was extracted from 10 ml samples of eight 150-ml cultures (4 containing 2 µg/ml AZM and 4 without AZM). In brief, bacteria were lysed in the presence of 10 ml killing buffer (Tris/HCL, 5 mM MgCl2, 2 mM NaN3, pH 7.5) and centrifuged at 4°C for 10 min. The aqueous phase was mixed carefully with 125 µl Saccharose/Na-Acetate (300 mM Saccharose, 10 mM NaAc, pH 4.5) and 125 µl SDS/ Na-Acetate (83 mM SDS, 10 mM NaAc, pH 4.5) was added. The suspension was heated to 65°C and 400 µl hot phenol was added. After 3 min of incubation at 65°C, the suspension was quickly cooled in liquid nitrogen and centrifuged for 10 min. The phenol extraction was repeated twice before the nucleic acids were pelleted with 40 µl 3M NaAc (pH 5.2) and 1 ml 90% ethanol at -20°C overnight, washed with 70% ethanol and treated with 40 U of DNaseI (Roche) in DNaseI buffer (20 mM NaAc, 10 mM MgCl2, 10 mM NaCl, pH 4.5) for 30 min at 37°C. After purification with RNeasy columns (Qiagen) the yield of total cellular RNA was determined by UV absorption. cDNA was synthesized from RNA pooled from two independent cultures and subsequently two GeneChips were hybridized for each culture condition (4 independent GeneChips in total). cDNA synthesis and biotin-ddUTP terminal labeling were performed in compliance with the manufacturer’s instructions for the P. aeruginosa GeneChip: Ten micrograms of total RNA were mixed with random primers (Invitrogen) and incubated for 10 min at 70°C. Then a cDNA reaction mix was added, containing 10 µl of 5 x first strand buffer, 0,1 µmol dithiothreitol, deoxynucleotide triphosphate (3 µl of a dNTP-mix, 10 mM each) and 50 U of SuperScript II (Reverse Transcriptase). This was followed by an incubation step for 10 min at 25°C, 60 min at 37°C, 60 min at 42°C, and 15 min at 72°C. RNA was degradated by the addition of 1 N NaOH (20 µl) and an incubation for 30 min at 65°C. Subsequently the reaction mix was neutralized by addition of 1 N HCl (20 µl). The cDNA was purified with QIAquick columns (Amersham Biosciences), quantified by UV absorption, fragmentized in one-for-all buffer with 0.5 U of DNase I (Amersham Pharmacia Biotech) per µg of cDNA for 10 min at 37°C and subsequently inactivated for 10 min at 98°C. The fragmentation of the cDNA (50-200-bp range) was checked by agarose gel electrophoresis. The fragmentized cDNA was labeled with the Enzo BioArray terminal labeling kit with biotin-ddUTP (Affymetrix). Keywords = aeruginosa Keywords = Azithromycin Keywords = macrolide antibiotics Keywords = quorum sensing Keywords = cystic fibrosis
