Murine neuroblastoma cell line Neuro2A was cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS at 37°C under 5% CO2 To induce neuronal differentiation, the cells were plated at a density of 3.8 x 10e3 cells/cm2. After 48 h, cells were harvested and RNA was extracted at time 0 h as undifferentiated control, 4-6 plates per replicate. Total RNAs were isolated from the Neuro2A cells using TRIZOL (Invitrogen) reagent. The poly (A)+ RNAs were isolated from the total RNAs using the uMACS mRNA Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's protocol. mRNAs (1 ug) were labeled with Cy5 dye using the BD PowerScript Fluorescent Labeling Kit (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's protocol (except for a partial modification of the priming procedure, in which we annealed 0.36 ug random nonamer at 42°C for 90 min). The labeled cDNAs were purified by using a QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA). After the cDNAs were mixed with 2 ug mouse Cot-1 DNA and 2 ug poly dA, they were precipitated by ethanol. The precipitant was dissolved in 40 ul hybridization buffer (50% formamide, 5 x SSC, 0.5% SDS), denatured at 100°C for 5 min, and incubated at 42°C for 15 min. Before hybridization, mKIAA oligonucleotide microarrays were treated with blocking buffer (1% BSA, 4 x SSC, 0.5% SDS) at 42°C for 45 min, washed twice with sterile water, and then immediately dried by blown air. Hybridization was carried out at 42°C for 16 h using the Array Booster (Advalytix AG, Brunnthal, Germany). After hybridization, the arrays were washed twice with 2 x SSC/0.1% SDS buffer at 60°C for 10 min and then twice with 0.2 x SSC/0.1% SDS buffer at 60°C for 10 min. After further soaking at RT in 0.05 x SSC buffer, the slides were immediately desiccated by blown air and scanned on the FLA-8000 Fluorescent Image Analyzer (Fujifilm, Tokyo, Japan). The fluorescence intensities were quantified by Phoretix Array software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).
