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Sample GSM8533 Query DataSets for GSM8533
Status Public on Sep 30, 2003
Title 5C-I
Sample type RNA
Source name retinal pigmented epithelium/choroid from 2 month old male
Organism Mus musculus
Extracted molecule total RNA
Description 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
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 microliter 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. 30 minute washes 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 50 micron 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
Lot batch = 1040052
Submission date Jul 21, 2003
Last update date Oct 28, 2005
Contact name Sharon A. Boylan
Phone 530-752-4935
Fax 530-752-2270
Organization name University of California, Davis
Department Biological Chemistry
Lab Vitreoretinal Research Lab
Street address 2403 Tupper Hall
City Davis
State/province CA
ZIP/Postal code 95616
Country USA
Platform ID GPL144
Series (1)
GSE565 mouse RPE/choroid age comparison - ATLAS arrays

Data table header descriptions
SIGNAL_RAW total spot intensity
BKD_RAW median intensity of blank areas of array
VALUE spot intensity minus background, multiplied by normalization coefficient

Data table
MA337 1 1 0
MA160 1 1 0
MA156 148 1 75.776
V115 62 1 31.744
V116 3 1 1.536
V117 12 1 6.144
V135 15 1 7.68
V369 1 1 0
V379 0 1 0
V391 5 1 2.56
V393 3 1 1.536
V283 1 1 0
V284 11 1 5.632
V048 6 1 3.072
V222 13 1 6.656
V260 2 1 1.024
V366 144 1 73.728
M372 1 1 0
M388 1 1 0
M316 0 1 0

Total number of rows: 1185

Table truncated, full table size 16 Kbytes.

Supplementary data files not provided

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