Micro-array signal data were log-normalized using global loess
Median Cy3 and Cy5 signal intensities per spot were determined using Array Vision software (Imaging Research, St. Catharines, Ontario, Canada).
Micro-array slides containing 21,997 oligos from the Sigma-Compugen Mouse oligonucleotide library were spotted at the micro-array department of University of Amsterdam. Labeled samples were hybridised overnight in 50% formamide, 10x SSC, 0.2% SDS at 42 degrees C.
1 ug of total lung RNA was amplified using the Amino Allyl MessageAmp aRNA kit (Ambion) and labelled using Cy3 and Cy5 (Amersham) for samples and the common reference, respectively, according to the manufacturer's instructions. For each array, treated samples from individual animals were used. For a common reference total RNA of the lungs of Balb/C mice, which underwent intratracheal instillation with either saline or PM suspended in saline, were pooled together.
During the acclimatization period of at least 4 days, as well as during exposure and recovery, the animals were housed in 0.2-m3 stainless steel and Lexan inhalation chambers in the RIVM inhalation facilities. Mice were exposed for 8 hours to either 0.8 ppm ozone or clean air.
Necropsy was performed at 4 hours post-exposure. At necropsy, animals were weighted and anaesthetized with a mixture of Ketamine (100 mg/ml) and Rompun (20 mg/ml) and saline in a ratio of 10:4:14. Injection of anaesthetic was performed i.p. 2 ml/kg body weight, resulting in a dose of 70 mg/kg body weight for Ketamine and 6 mg/kg body weight for Rompun. Animals were sacrificed by exsanguination via the abdominal aorta. Saline perfusion of the lungs was performed via the right cardiac ventricle. Lungs were lavaged (three in- and out lavages using same fluid) with a volume of saline corresponding with 40 mL/kg body weight at 37 C. A second lavage was performed with an amount of saline which corresponds with the recovery volume of the first lavage. RNA was isolated from frozen, weighted left lung lobes (stored in RNAlater RNA Stabilization Reagent, Qiagen) using Trizol reagent (Invitrogen) and RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. Total RNA was treated with RNAse-Free DNase (Qiagen) to eliminate contaminating genomic DNA.
Csb+/- and Csb-/- mice (10-14 weeks old, 20-30 g) were bred at the National Institute for Public Health and the Environment (RIVM).
Arrays were scanned at two wavelengths using a ScanArray 4000XL microarray scanner (Perkin-Elmer)