NF-κB target gene microarray in HAECs in response to Pseudomonas aeruginosa

An NF-κB target gene array (Panomics, Inc., Redwood City, CA) was performed to profile the PAO1-dependent expression pattern of 111 NF-kB-regulated genes in primary human airway epithelial cells (HAECs). A small scale microarray was carried out using NF-κB target gene array kit (Panomics, Inc., Redwood City, CA). A long sense-strand oligonucleotide for each of the 111 human genes that have been previously shown to be regulated by NF-KB signaling pathway was spotted in duplicate on the nitrocellulose membrane. Biotinylated DNA was spotted along the right and bottom sides of the array membrane as control. The arrays were performed according to the manufacturer’s instructions. Keywords: cellular response to bacteria In brief, HAECs were stimulated for 18 hours with the heat-killed bacteria PAO1 (multiplicity of infection: 100:1). Control experiment used PBS instead of bacteria. mRNA of HAECs was isolated using biotin-labeled oligo(dT)20 and streptavidin-conjugated magnetic particles (Roche, Germany). 500 ng mRNA was used to prepare biotin-labeled cDNA probes through incorporation of biotin-dUTP into cDNA via reverse transcription reactions. Each biotin-labeled cDNA probe was hybridized to an array membrane at 42°C overnight in a hybridization incubator (Fisher Scientific, Pittsburgh, PA). The membrane was washed and subjected to further incubations with blocking buffer and subsequent streptavidin-conjugated HRP. The membrane was developed through chemiluminescence reactions and detected immediately by 1 min exposure to Hyperfilm ECL X-ray films (Amersham, Piscataway, NJ). The dots on the array were quantified through densitometric analysis using GeneTools (Syngene, Frederick, MD) and the value of each dot was determined as D = (signal value – background value)/mean value of biotinylated DNA dots. Ratio (Dbacteria: Dcontrol) was used to determine the effect of bacteria stimulation on the gene regulation. Ratio>1 indicated an up-regulation of the gene. The experiments include two samples, one PBS control sample and one PAO1-stimulated sample.