|Application:||Gene expression microarray analysis|
|Number of samples:||18|
|Release date:||Nov 29 2010|
|Last update date:||Aug 23 2018|
|Diseases:||Lacrimal Apparatus Diseases|
|Dataset link||Hypothalamic transcriptome plasticity in two rodent species reveals divergent differential gene expression but conserved pathways|
Animals. Adult male C57BL/6 mice (Harlan Sera-Lab, Loughborough, UK) were group housed (4 per cage) under controlled temperature (21+ 2ºC) and diurnal light conditions (14-h light, 10-h dark, lights on at 05.00). Food and water were available ad libitum until the experiment commenced. Complete fluid deprivation was imposed for 48 hours starting at 11.00 a.m. Control animals maintained free access to drinking water, and both groups had access to standard laboratory rodent chow. Experiments on adult male rats described previously (27). All procedures were conducted in strict accordance with the Animal Scientific Procedures Act (1986), UK, and were approved by the local University of Bristol Ethical Review Process. Tissue collection. Mice were killed using cervical dislocation and the brain was carefully removed from the cranium and snap frozen using powdered dry ice and stored at -80oC for no more than 14 days. Sections of brain (14μm) were cut using an RNase free cryostat and mounted onto RNase free membrane coated glass slides (P.A.L.M. Membrane slides; P.A.L.M. Microlaser Technologies). Immediately after sectioning, frozen sections were thawed and fixed (30s; in 95% [v/v] EtOH), rehydrated (30s in each of 75% [v/v] and 50% [v/v] EtOH) before being stained (60s 1% [v/v] cresyl violet). Sections were then dehydrated in a graded EtOH series (30s in each of 50% [v/v], 75% [v/v] and 95% [v/v]. then 2x 30s in 100% [v/v]). Laser microdissection was performed using a P.A.L.M. MicrolaserSystem (P.A.L.M. Microlaser Technologies). The SON was identified with reference to Franklin and Paxinos (28) and the tissue from each animal was independently pooled into collection vials containing RNAlater® (Ambion, Huntingdon, UK). A single operative carried out all dissections. Total RNA was isolated without delay (within 24h) according to standard procedures that accompany the Ambion RNAqueous MicroKit (Ambion). Microarray analysis. Separate microarrays (n=4) were probed using independently generated target. For each completely independent replicate, tissue from 1 mouse was used for RNA extraction.