strain: CD-1 gender: male age: 10 weeks source tissue: brain
Treatment protocol
Brains were collected, dissected in slices of 25µm and mounted to Superfrost microscope slides (Menzel, Braunschweig, Germany) or onto LMD6000 metallic frame slides covered with a membrane of polyethylene terephthalate (Leica Microsystems Deutschland, Bensheim, Germany) in a cryostat (Microm MH50, Microm, Walldorf, Germany) from rostral to caudal. Only brain slices containing the brain areas of interest were sampled, including the anterior part of the cingulate cortex (Cg), the hypothalamic paraventricular nucleus (PVN), the anterior dentate gyrus (DG), the basolateral (BLA) and the central amygdala (CeA). For the DG, the same coordinates as for the PVN were applied. To cover the whole area of interest an overall depth of 400µm per region was chosen. While sampling the CG, BLA and CeA four brain slices were mounted onto an LMD6000 metallic frame slide (Leica Microsystems), the following four to Superfrost slides (Menzel). This procedure was repeated for the next eight slices. For the region containing the PVN and DG only two slices were mounted to one slide always alternating the LMD6000 frame and the Superfrost microscope slides. Only the LMD6000 slides were used for further processing and stored at -80°C. Right before laser-microdissection, brain slices on the LMD6000 frame slides were stained with cresyl violet applying a modified staining protocol that included 1.5min staining in cresyl violet, followed by washing in 70% and 96% ethanol for 20s each and in isopropanol for 5min. The slides were refrozen and processed at the laser-microdissection microscope (AS LMD, Leica). For the dissection procedure of the required brain areas, a magnification of 100x was chosen with laser power between 80-100% and speed varying between 1 and 4. Cut-out brain areas were captured in the caps of 0.2ml PCR soft tubes (Biozym Scientific, Hessisch Oldendorf, Germany) and cooled on dry ice immediately after completion of one brain region. Pictures of the processed brain slices were acquired by means of the IM1000 software. Microm, Walldorf, Germany) from rostral to caudal.
Extracted molecule
total RNA
Extraction protocol
From all samples, total RNA was extracted individually (one sample per mouse and brain region) in presterilized 1.5ml Safelock tubes (Eppendorf, Hamburg, Germany) using a TRIzol (Invitrogen, Karlsruhe, Germany) chloroform standard protocol. After tissue homogenization in 300µl of TRIzol by pipetting, 1µl linear acrylamide (5mg/ml, Ambion, Austin, TX) and 60µl of chloroform (Carl Roth, Karlsruhe, Germany) were added and the samples vortexed. Centrifugation for 5min at 18°C and 13,000rpm followed, then RNA was precipitated with 180µl isopropanol (Carl Roth) overnight at -20°C, centrifuged at 4°C and 13,000rpm for 30min and washed twice in 500µl 70% ethanol (Carl Roth) with centrifugation steps at 4°C and 13,000rpm of 10min in between. Following the last centrifugation step, all remaining liquid was removed with a pipet, pellets were dried in an incubator for 15min at 45°C and resolved in 13µl of autoclaved bidistilled water.
Label
biotin, Cy3
Label protocol
RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit (Ambion) with only one round of in vitro transcription, where biotinylated uracyl bases were built in to the newly synthesized aRNA. 5µg per sample were required for loading to the microarray slides. Correct quantification was ensured by measurement of optic density in a NanoPhotometer (Implen, Munich, Germany) and additional analysis on agarose gel. Samples not fulfilling all criteria of homogeneity (inadequate concentrations, too many small but few larger aRNA fragments) were excluded from further analysis.
Hybridization protocol
Each microarray slide had the capacity for six samples, same brain regions were hybridized in the same batch comparing a maximum of six individual mice of each breeding line. Reagents and material were provided by Illumina, the procedure was strictly conducted according to the manufacturer’s protocol. In brief, each sample was mixed with hybridization buffer, loaded onto the designated array field and the slides were put into hybridization chambers and incubated for 17h in incubation chambers provided by Illumina. Arrays were washed in several steps, incubated with Cy3, washed several times again and dried by centrifugation.
Scan protocol
Fluorescence signals were detected on a BeadStation scanner (Illumina) and analyzed by the BeadStudio (Illumina) software. The manufacturer's built-in controls have been analyzed including hybridization controls and sample dependent parameters. Only microarrays fulfilling Illumina's recommendations for quality control have been used for further evaluation.
Description
Biological replicate of NAB 1
Data processing
Illumina BeadStudio gene expression results were further analyzed using R-packages, based on ‘beadarray’ system as described by Dunning et al. (2007), which simplifies the comparison between a high number of arrays. First, pair wise box plots were generated to compare mean expression within each line and brain region. Normalization for expression values has been applied to all samples with the ‘QSpline’ function. Each sample has been clustered to ensure that each brain region per line shows similar expression patterns using the ‘hclust’ function. Three samples from different brain regions have been identified as inadequate during the scan process and have therefore been excluded from further analysis. For differential expression analysis, functions of the ‘LIMMA’ package have been applied on log2-transformed values. The resulting matrix has been used for all subsequent analyses. Significantly regulated genes were ranked using an empirical BAYES method implemented in the LIMMA R-package (Lonnstedt and Speed, 2002; Smyth, 2004).
