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Sample GSM8534 Query DataSets for GSM8534
Status Public on Sep 30, 2003
Title 12C-I
Sample type RNA
 
Source name retinal pigmented epithelium/choroid from 24 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.
Keywords = retinal pigmented epithelium
Keywords = aging
Keywords = choroid
Lot batch = 1040052
 
Submission date Jul 21, 2003
Last update date Oct 28, 2005
Contact name Sharon A. Boylan
E-mail(s) saboylan@ucdavis.edu
Phone 530-752-4935
Fax 530-752-2270
URL http://biochem.ucdavis.edu/labs/Hjelmeland/index.html
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
ID_REF
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
ID_REF SIGNAL_RAW BKD_RAW VALUE
MA337 1 1 0
MA160 1 1 0
MA156 156 1 92.82
V115 135 1 80.325
V116 1 1 0
V117 4 1 2.38
V135 12 1 7.14
V369 0 1 0
V379 1 1 0
V391 2 1 1.19
V393 0 1 0
V283 1 1 0
V284 9 1 5.355
V048 5 1 2.975
V222 10 1 5.95
V260 2 1 1.19
V366 155 1 92.225
M372 2 1 1.19
M388 0 1 0
M316 1 1 0

Total number of rows: 1185

Table truncated, full table size 15 Kbytes.




Supplementary data files not provided

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