Gene expression beadchip array

An aliquot of 150 ng of high-quality total RNA from each sample was used for mRNA expression profiling. Samples were analyzed using Illumina® Gene Expression BeadChip Array technology (Illumina, Inc., San Diego, CA). Reverse transcription for synthesis of the cDNA strand, followed by a single in vitro transcription (IVT) amplification, that incorporates biotin-labeled nucleotides, were performed with Illumina® TotalPrep™ -96 RNA Amplification Kit (Ambion, Austin, TX). 750 ng of the biotin-labeled IVT product (cRNA) was hybridized to HumanHT-12v4_BeadChip (Illumina, Inc., San Diego, CA) for 16 h, followed by washing, blocking, and streptavidin-Cy3 staining according to the Whole Genome Gene Expression Direct Hybridization protocol (Illumina, Inc., San Diego, CA). The arrays were scanned using HiScanSQ System and obtained decoded images were analyzed by GenomeStudio™ Gene Expression Module—an integrated platform for the data visualization and analysis (Illumina, Inc., San Diego, CA).

RNA was isolated from the whole hippocampus of mice using the RNeasy Mini Kit (Qiagen) according to the manufacturer instructions and quantified using a spectrophotometer (model ND-1000, NanoDrop Technologies). RNA quality was analyzed by determining the RNA Integrity Number (RIN) using a bioanalyzer (model 2100, Agilent Technologies). High-quality RNA (with RIN of at least 6) was used to perform an expression profile of mRNA using the Gene Expression BeadChip Array (Illumina) according to the manufacturer guidelines. In brief, the cRNA library was first generated from mRNA and amplified using an RNA amplification kit (Illumina TotalPrep-96, Ambion). Biotin-linked nucleotides were hybridized to the HumanHT-12-v4-BeadChip (Illumina) for 16 h, sequentially followed by washing, blocking, and staining with Streptavidine-Cy3 following the Expression Direct Hybridization protocol (Illumina). Arrays were scanned using the HiScanSQ System, and images were analyzed by GenomeStudio Gene Expression Module (Illumina). Data were analyzed using one-way ANOVA at p < 0.05. Heatmap visualization of the expression data were performed using the heatmap.2 function from the gplots package (Warnes et al., 2005 ).