I. Exp Design 1. Type of experiment: Young vs Aged comparison 2. Experimental factors: 2 month old mouse vs 24 month old mouse 3. How many hybridizations in exp: 12 4. If a common reference used for all the hybs: No 5. Quality Control steps: three independent replicates for each age 6. Description – Young mice were compared to old mice to determine gene changes that occur with aging in the mouse retinal pigmented epithelium/choroid of the eye
II. Samples Used, extract prep, and labeling 1. Biosource: Mus musculus strain C57BL/6 from National Institute on Aging Old mice available from National Institute on Aging, young mice from Jackson Labs Male mice, 2 and 24 months old, used only a portion of the eye, the retinal pigmented epithelium in combination with the choroid (RPE/choroid) with no tissue from other parts of the eye 2. Manipulations: Animals were allowed to acclimate to our facilities for two weeks prior to death by cervical dislocation- no treatments were performed. Eyes were removed from each mouse, the anterior segments were removed with a circumferential incision, and posterior segments were then separated from the whole retina. The pigmented layer including RPE/choroid was scraped gently from the sclera as a sheet in less than 50 ml of RNAlater buffer (QIAgen, Valencia, CA). 3. Extract preparation: RPE/choroid tissue was placed into lysis buffer RLT (Qiagen) and homogenized using needle and syringe followed by QIAshredder (Qiagen). Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer’s instructions. DNase treatment (Qiagen) was used to remove any DNA contaminants. The Atlas SMART PCR system from Clontech was used to amplify the RNA using PCR following the manufacturer’s instructions starting from 500 ng total RNA. 4. Labeling protocol: the SMART cDNA probe labeling kit from Clontech was used to prepare 33P-labeled cDNA starting with 500 ng of purified cDNA resulting from amplification. 5. No external controls were added.
III. Hybridization procedures and parameters 1. Sample , array type, batch and serial # used 2. Hybridization protocol: Each hybridization reaction contained 500 ng labeled cDNA (denatured at 100ºC) in 10 ml ExpressHyb (for a single array). Arrays were blocked with SMART Blocking solution. Hybridization was done in 10 ml volume at 65ºC overnight using a hybridization oven set at 5-7rpm. Wash at 65ºC with 2XSSC,1%SDS (3X); 0.5XSSC,0.5%SDS (1X); and 0.33XSSC,0.5%SDS (1X).
IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3-5 days and scanned at 50mm resolution using the Storm 860 (Molecular Dynamics, Sunnyvale, CA). Gel images from the phosphorimager were exported to the AtlasImage 1.5 software (Clontech) for analysis. 3. Data processing: A gene was identified as expressed if its background subtracted intensity was greater than 1.4 times background. The data were normalized using a simple global scaling procedure, and the Significance Analysis of Microarrays (SAM, version 1.15) program was used to determine significant differential gene expression. Keywords = retinal pigmented epithelium, aging, choroid
