Limbic Brain Transcriptome Analysis

DNA microarray analysis was conducted on isolated RNAs extracted from the limbic area of mice brains as reported previously [10 (link)]. Double-stranded cDNA was synthesized from 100 ng of total RNA with the GeneAtlas 3´ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Biotin-labeled amplified RNA (aRNA) was synthesized by in vitro transcription using the GeneChip 3´ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Briefly, purified aRNA was fragmented using the GeneAtlas 3´ IVT Express Kit and hybridized for 16 h at 45°C using the GeneChip MG-430 PM microarray (Affymetrix Inc., Santa Clara, CA, USA). The chip was washed and stained in the Gene Atlas Fluidics Station 400 (Affymetrix Inc., Santa Clara, CA, USA), and the resulting image was scanned using the GeneAtlas Imaging Station (Affymetrix Inc., Santa Clara, CA, USA). Data analysis was performed using the Affymetrix Expression Console Software version and Visualization and Integrated Discovery (DAVID) software version 6.8 (National Institute of Allergy and Infectious Diseases (NIAID). Compared with the control (vehicle-treated group), fold-changes in the expression of genes in the imipramine- or EEA-treated groups were calculated and converted to linear data.