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 107 mouse genes that have been previously shown to be regulated by NFkB 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. Briefly, 20 micrograms of Total RNA extracted from ES cells grown in the presence or absence of Leukemia Inhibitory Factor were 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. 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 10 seconds exposures to Kodak BioMax XAR films (Sigma, Dorset, UK). The dots of the array were quantified through densitometric analysis using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda,Maryland, USA, http://rsb.info.nih.gov/ij). The 2 spots intensities acquired for each gene were averaged to obtain the VALUE. Empty spots were used as background values. Ratio (minus:plus LIF) was used to determine the effect of ES cell differentiation on the gene regulation.
