A small scale cDNA 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-B 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 of the array. The arrays were performed according to the manufacturer’s instructions. In brief, HAECs were stimulated for 18 hours with the heat-killed bacteria PAO1 or PAK (multiplicity of infection: 100:1). Cells incubated with medium were used as control. mRNA of HAECs was isolated using biotin-labeled oligo(dT)20 and streptavidin-conjugated magnetic particles (Roche, Germany). 500 ng mRNA of each sample 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 after hybridization and subject to incubations with blocking buffer and subsequent streptavidin-conjugated horseradish peroxidase (HRP). The detection was carried out through chemiluminescence reactions and 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 is determined in the equation D = (raw 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.
