rats were obtained from the National Institute of Aging rat colony
Treatment protocol
animals were trained in the water maze. SIT = control rats stayed in cages, YOKE=swam with no platform, VIS= were trained with visible platform, HID= animals were trained in the hidden version of the paradigm
Growth protocol
Fisher-344 male rats, 3 and 24 month old, were housed (2 per cage) in a 12:12 light-dark cycle with ad libitum access to food and water. Behavioral testing occurred during the light phase of the cycle.
Extracted molecule
total RNA
Extraction protocol
Animals were sacrificed 24 hours after the last behavioral trial by decapitation, following a lethal dose of pentobarbital (100 mg/kg), and the brains were extracted from the skull and washed with ice-cold PBS. The brains were sectioned into 1 mm slices and placed in RNAlater (Ambion). The brain slices were stored at -20°C until ready for further dissection and RNA isolation. The RNAlater gave the tissue a “fixed” consistency and allowed for clear visualization of the hippocampal cellular layers. Three main anatomical areas of the hippocampus were dissected. Using a 300 µm micro punch (Electron Microscopy Sciences), the main cell layer of the CA1 region from each animal was dissected out and placed in tubes containing 50 µl of RNAlater. RNA was isolated using the Qiagen RNeasy protocol (Qiagen). The tissue was homogenized in 400 µl of RLT buffer and 1% mercaptoethanol, and treated with 200 µg/ml proteinase K for 10 minutes at 55ºC. After RNA isolation with the Qiagen RNeasy kit we assessed the quality of the RNA using an Agilent 2100 bioanalyzer using the RNA 6000 Nanochips assay kit (Agilent Technologies). The RNA was stored at -80°C until further use.
Label
biotin
Label protocol
two rounds of RNA amplification were performed following the Affymetrix Small Sample Target Labeling version I protocol, which consists of two cycles of cDNA synthesis. (The detailed protocol can be obtained at the Affymetrix website: http://www.affymetrix.com/index.affx).
Hybridization protocol
standard Affymetrix procedures
Scan protocol
standard Affymetrix procedures
Description
A HID U_B015 The first letter refers to the age of the animal (Y= young, 3month old rat; or A=aged, 24 month old rat). The second word refers to the treatment, as described in sample treatment protocol. The third descriptor, eg, B003, B refers to the region of the hippocampus (CA1 region) animal number 3. In addition, aged animals who underwent the hidden platform (HID), can either be learning impaired (I) or hidden unimpaired (U)
