Animals were sacrificed by cervical dislocation and brain regions were immediately dissected while being kept cool with partially frozen ASCF (124 mM NaCl, 26 mM NaHCO3, 10 mM Glucose, 3.3 mM KCl, 1 mM NaHPO4, 2 mM CaCl2, 2 mM MgCl2). Tissue was then frozen using liquid nitrogen (Praxair).
All animal procedures were approved by the Animal Management Committee of Mount Sinai Hospital and complied with the requirements of the Province of Ontario Animals for Research Act 1971 and the Canadian Council on Animal Care. Groups of 3-5 littermates were housed by sex in polycarbonate cages, and given ad libitum sterile food (Purina mouse chow) and water. The vivarium was maintained under a controlled temperature (21ºC ± 1ºC), and humidity (50-60%), with a 12-h diurnal cycle (lights on: 0700-1900). Animals with the SrrY269* mutation used in experiments contained >99.6% of the C57BL/6J genome. The microarray study was conducted on experimentally naïve male mice that were 9 weeks of age.
Dissected tissue from whole brain, hippocampus, frontal cortex, and cerebellum was homogenized in 1mL of Trizol (Invitrogen) using a Polytron homogenizer (PRO Scientific). A 5-min incubation at room temperature preceded and followed the addition of 200 μL of chloroform (Sigma). Samples were centrifuged at 12,000 × g for 15 min at 4°C and the aqueous phase was collected. Total RNA was precipitated from the aqueous phase using 500 μL of isopropyl alcohol (Sigma), and samples were then incubated at room temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4°C. The RNA pellet was washed with 1 mL of 75% ethanol, centrifuged at 7,500 × g for 5 min at 4°C, and dissolved in 100 μL of DEPC-treated water (Applied Biosystems). For the microarray experiment, additional purification of total RNA was completed with the Absolutely RNeasy RT-PCR Miniprep kit (Strategene). RNA yield was quantified by UV spectrophotometry (NanoDrop ND-1000; Thermo Fisher Scientific) and RNA integrity was verified using the Agilent Bioanalyzer 2100 system (Agilent Technologies).
Biotinylated and amplified cRNA was generated using Illumina® TotalPrep RNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX).
Biotinylated and amplified cRNA was generated using Illumina® TotalPrep RNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX). Labeled cRNA samples (750 ng) were then hybridized to MouseRef-8 v2.0 Expression BeadChips (Illumina, Inc., San Diego, CA) in accordance with Illumina’s standard protocols for hybridization, staining, and data acquisition.
BeadChips were imaged using the Illumina BeadArray Reader (Infinium I FastScan scanner protocol), a two-channel 0.8 μm resolution confocal laser scanner. The BeadScan software was used for the identification of bead positions and the extraction of raw-data.
Illumina data was pre-processed separately and identically for each tissue. All pre-processing was performed in the R statistical environment (v2.7.2) using the BioConductor open-source library (Gentleman RC et al. Genome Biol 2004). Raw array data (TIFF files) were first processed using the beadarray package (v1.8.0) (Dunning MJ et al. Bioinformatics 2007). The raw data was subsequently background-corrected using the non-linear Edwards model (Edwards D Bioinformatics 2003). Following careful quality assessment at the bead and gene level, no arrays were excluded. The background-corrected data was summarized in normal space using the standard Illumina method. The summarized data was transformed using the vst method as implemented in the lumi package (v1.6.2) (Lin SM et al. Nucleic Acids Res 2008), and then normalized using quantile splines (Workman C et al. Genome Biol 2002).