Pseudomonas aeruginosa PAO1 Immortalised, Cystic Fibrosis Tracheal Epithelial (CFTE) cells, Delta F508 mutation of CFTR (Gruenert et al. 2004) For epithelial cell infection studies, cells were washed twice with sterile phosphate-buffered saline (PBS), detached and seeded in antibiotic-free media into newly-coated plastic vessels 24h prior to infection so as to achieve 80% confluency at the time of infection. Bacterial strains were cultured aerobically for 16-18 h in the described cell culture medium without antibiotics (infection medium) at 37°C. Bacteria were sub-cultured in infection medium for an additional 4 h using the growth conditions described so that bacterial strains were in the exponential phase of growth. Following three rounds of centrifugation and PBS washing to remove extracellular components, bacterial densities were adjusted so as to infect cells at a multiplicity of infection (MOI) of 50:1. Serial dilutions were plated onto Luria-Bertani agar to confirm the MOI used. At the indicated times postinfection, cells which were already detached from the plastic were collected by centrifugation and those cells remaining attached were treated with trypsin-EDTA prior to collection by centrifugation. Harvested cells were immediately frozen at -70°C for subsequent RNA isolation. RNA isolation and microarray sample preparation Biological samples from two independent infection experiments were used in two independent microarray experiments as outlined below. Total RNA was isolated from epithelial cells using an RNeasy Kit (Qiagen) according to manufacturer’s instructions. Genomic DNA was removed using DNA-free (Ambion) and confirmed by PCR to be free of DNA, prior to cDNA synthesis. Isolated RNA was used in the preparation of fragmented cRNA for microarray hybridization according to the manufacturer’s protocol (Affymetrix). Briefly, 8.5µg of RNA was converted to double stranded cDNA by reverse transcription, using a Superscript cDNA synthesis kit (Invitrogen), with an oligo dT primer containing a T7 RNA polymerase site added 3’ of the poly (T) (Affymetrix). After second strand synthesis, cDNA was converted to biotin-labelled cRNA by in vitro transcription (Enzo). The labelled cRNA was purified using Genechip clean up module (Qiagen). Total cRNA quality and yield was measured using a GeneQuant spectrophotometer and 20 µg of cRNA was fragmented at 94ºC for 35 mins in fragmentation buffer (40 mM tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). Full length and fragmented cRNA sample integrity was assessed by electrophoresis on a 1% agarose-TBE gel visualized by staining with SyBr green. Fragmented cRNA was sent to MRC geneservice (Cambridge, UK) for assessment of RNA quality using a Bioanalyzer (Agilent) before hybridization to Human Genome U133A array (Affymetrix). The arrays were washed and stained in a fluidics station and scanned according to the manufacturer’s protocols (Affymetrix).
Extracted molecule
total RNA
Label
Biotin
Description
Briefly, 8.5µg of RNA was converted to double stranded cDNA by reverse transcription, using a Superscript cDNA synthesis kit (Invitrogen), with an oligo dT primer containing a T7 RNA polymerase site added 3’ of the poly (T) (Affymetrix). After second strand synthesis, cDNA was converted to biotin-labelled cRNA by in vitro transcription (Enzo). The labelled cRNA was purified using Genechip clean up module (Qiagen). Total cRNA quality and yield was measured using a GeneQuant spectrophotometer and 20 µg of cRNA was fragmented at 94ºC for 35 mins in fragmentation buffer (40 mM tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). Full length and fragmented cRNA sample integrity was assessed by electrophoresis on a 1% agarose-TBE gel visualized by staining with SyBr green. Fragmented cRNA was sent to MRC geneservice (Cambridge, UK) for assessment of RNA quality using a Bioanalyzer (Agilent) before hybridization and scanning. The arrays were washed and stained in a fluidics station and scanned according to the manufacturer’s protocols (Affymetrix).
