ny possibility of wrong annotation the majority of the dif ferentially expressed genes, in particular those of major interest were sequenced. All data are deposited at the GEO server. Ontology Groups Classification and Molecular Pathways Building To classify and characterize the differentially expressed genes that resulted from the microarray analysis the Gene Ontology data annotation was used. In addition, the text mining program Pathway Stu dio 5. 0 was used for the identification of pathways linking the regulated genes. Downstream targets of the genes regulated 4 h after stress were looked for in the group of genes regulated 8 h after stress and upstream regulators of the latter in the former group. The main criteria applied for the final selection of the pathways were, 1.

the last step of the pathway has to indicate molecular synthesis expression so as to validate the expression change described on the microarray result, 2. confirmation of the annotation of the selected genes by sequencing and 3. validation of the literature references used by the program. In situ Hybridisation Dacomitinib 18 um thin cryostat sections prepared from frozen stored brains were thaw mounted on poly L Lysine coated slides, dried and kept at 80 C. UTP labelled riboprobes for amyloid b precursor protein and guanine nucleotide binding protein, alpha inhibiting 2 genes were prepared by in vitro transcription from corresponding cDNA clones. The sections were dried, fixed in 4% paraformaldehyde, washed in PBS and subjected to acetylation using 0. 25% acetic anhydride in 0. 1 M triethanolamine HCL, pH 8.

0. After subsequent dehydration in increasing concentra tions of ethanol, brain sections were saturated with 100 ul of hybridisation buffer containing 2. 5 �� 106 cpm 35 S labelled riboprobe. Brain sections were incubated overnight at 62 C or 58 C. Then, the sections were rinsed in 4�� SSC, treated with RNAse A and washed in increasingly strin gent SSC solutions at room temperature. Finally sections were washed in 0. 1�� SSC for 1 h at 67 C or 64 C and dehydrated through increasing con centrations of ethanol. Autoradiography was on Biomax MR film for 3 5 days. The autoradiographs were digitised, and relative expres sion levels were determined by computer assisted optical densitometry. The average value of 4 6 measurements was calculated from each animal.

Quantitative PCR A total of 200 ng of amplified RNA from the first ampli fication round of the microarray analysis was reverse transcribed with Superscript II using random hexamer primers according to the manufacturers protocol. For quality control, a small aliquot of each cDNA was analyzed on an agarose gel. parison. Relative gene expression was determined by the 2 CT method using the real PCR efficiency calcu lated from an external standard curve. Cp were normal ized to the housekeeping genes GAPDH, HPRT1, POLR2B or RPL13A Fold regulation values were calcu lated relative to the expression mean of basal mice. The calculation Cp Cp was