NFkB target gene microarray during LIF-withdrawal differentiation of mouse embryonic stem cells

An NFkB target gene array (Panomics, Inc., Redwood City, CA) was performed to profile the expression pattern of 107 NFkB-regulated genes in mouse embryonic stem cells. A small scale microarray was carried out using NFkB target gene array kit (Panomics, Inc., Redwood City, CA). A long sense-strand oligonucleotide for each of the 107 human 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. The arrays were performed according to the manufacturer’s instructions. Keywords: embryonic stem cell differentiation CCE1.19 embryonic stem cells were plated on gelatinised plates at 2000 cells/cm2 and grown for 4 days in the presence or absence of Leukemia Inhibitory Factor (LIF). Medium was changed every other day. Then, total RNA was extracted using Tri-reagent (Helena Biosciences). 20 ug of RNA 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.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 10 seconds exposures to Kodac BioMax XAR film (Kodak, 8532665). 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 signals were used as background and intensities substracted from the VALUE. The experiments include two samples, one with cells grown in the presence of LIF (control) and one of cells grown in the absence of LIF (experimental).