Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 µM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 °C and 300 rpm. The aerobic culture was grown to an OD578 of 0.3 and then 130 ml were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions.
Extracted molecule
total RNA
Extraction protocol
25 ml of the culture were cooled down with crushed ice, harvested and subjected to total RNA isolation by a modified hot phenol method (Aiba et al., 1981; von Gabain et al., 1983). The obtained RNA was treated with RNase-free DNase I and further purified by RNeasy columns (Qiagen, Hilden, Germany). The integrity of total RNA as well as the transcription of anaerobic marker genes in the harvested cells was confirmed by Northern Blot analysis.
References: Aiba, H., S. Adhya & B. de Crombrugghe, (1981) Evidence for two gal promoters in intact Escherichia coli. J Biol Chem 256: 11905-11910. von Gabain, A., J. G. Belasco, J. L. Schottel, A. C. Chang & S. N. Cohen, (1983) Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A 80: 653-657.
Label
biotin
Label protocol
cDNA was synthesized from RNA pooled from three independent cultures and subsequently hybridyzed on one GeneChip. The cDNA synthesis, fragmentation and labeling were performed according to protocols for the Affymetrix Pseudomonas GeneChip® (Santa Clara, California) with the following modification: 0.6 U DNase I were used per µg cDNA for fragmentation to yield the desired cDNA size range of 50 - 200 bases as confirmed by gel electrophoresis. This was verified by separating 200 ng of fragmented cDNA on a 15 % acrylamide gel and staining with SYBRgold (Molecular Probes, Eugene, United States).
Hybridization protocol
The GeneChip was hybridized at the Affymetrix core facility at the Helmholtz Centre for Infection Research in Braunschweig for 16 h at 50°C and 60 rpm using an GeneChip Hybridisation oven 640. Washing was done using an Affymetrix Fluidics Station 400.
Scan protocol
Scanning was done at the Affymetrix core facility at the Helmholtz Centre for Infection Research in Braunschweig using an HP GeneArray scanner for quantification with Affymetrix Microarray Suite Version 5.0.
Description
RNA was pooled from three independent cultures prior to cDNA synthesis. cDNA from three independent cultures was hybridized to one microarray.
Data processing
Raw data obtained from the Affymetrix GeneArray Scanner (CEL files) were preprocessed with the Bioconductor software framework. Expression values were computed with the Robust Multichip Average (RMA) method (Irizarry et al. 2003, Irizarry et al. 2003a) from the perfect match intensities, using quantile normalization and median polish as summarization method.
References: Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U. and Speed, T.P. (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics, 4, 249-264 Irizarry, R.A., Bolstad, B.M., Collin, F., Cope, L.M., Hobbs, B. and Speed, T.P. (2003a). Summaries of Affymetrix GeneChip probe level data., Nucleic Acids Res. , 31, e15
