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Sample GSM115965 Query DataSets for GSM115965
Status Public on Jun 22, 2007
Title Retinal pigment epithelium
Sample type RNA
Source name Primary cultured cells of retinal pigment epithelium
Organism Mus musculus
Characteristics Retinal pigment epithelium (Primary cultured cell)
Treatment protocol The retinal tissue was triturated to make a single cell suspension, and then re-suspended in the medium.
Growth protocol Primary cultures of pigmented epithelial cells from the retinal PE (RPE) were cultured in DMEM complete medium composed of DMEM and 20% FBS. The medium of ocular PE was changed to serum free medium, with a cellular RNA extraction then performed after an overnight culture.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using TRIZOL Reagent(Invitrogen) and used as starting material for the cDNA preparation. RNA was purified from total cellular RNA by Nucleospin RNA II (Macherey-Nagel, Inc, Germany) as outlined by the manufacturer. Integrity of total RNA was assessed qualitatively on the Agilent 2100 Bioanalizer (Agilent Technologies).
Label biotin
Label protocol 1 micro gram of total RNA was reverse transcribed using the cDNA synthesis system of MessageAmp aRNA Kit (Ambion). The oligonucleotide used for priming was 5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3. Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed using the 3'-Amplification Reagents for IVT Labeling (Affymetrix). The cRNA was cleaned using RNeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc.
Hybridization protocol 10 migro grams of fragmented cRNA were hybridised (45 degrees Celsius, 16 hours). Hybridization was controled adding cRNA cocktail (BioC, BioD, Cre) mixed as described in previous versions of the Expression Analysis Technical Manual. Washing and staining was performed in a Fluidics Station 450 (Affymetrix) using the protocol EukGE-WS2v5.
Scan protocol Scanning was performed in an Affymetrix GeneChip scanner 3000.
Description The primary RPE cultures were found to be greater than 95% cytokeratin positive after 14 days of incubation.
Data processing The data analysis were performed using the Affymetrix GeneChip Operating Software v1.3. using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of array was arbitrarily set to 100.
Submission date Jun 23, 2006
Last update date Jun 26, 2006
Contact name Yuri Futagami
Organization name Tokyo Medical and Dental University
Department Ophthalmology
Street address 1-5-45 Yushima Bunkyoku
City Tokyo
ZIP/Postal code 113-8519
Country Japan
Platform ID GPL1261
Series (1)
GSE5134 Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells.

Data table header descriptions
VALUE MAS5-calculated Signal intensity
ABS_CALL the call in an absolute analysis that indicates if the transcript was present (P), absent (A), marginal (M), or no call (NC)
DETECTION P-VALUE 'detection p-value', p-value that indicates the significance level of the detection call

Data table
AFFX-BioB-5_at 2.1 A 0.868639
AFFX-BioB-M_at 1.4 A 0.978098
AFFX-BioB-3_at 0.6 A 0.995519
AFFX-BioC-5_at 484.4 P 0.000044
AFFX-BioC-3_at 448.9 P 0.000044
AFFX-BioDn-5_at 818.6 P 0.000044
AFFX-BioDn-3_at 1943 P 0.000044
AFFX-CreX-5_at 5679 P 0.000044
AFFX-CreX-3_at 7111.7 P 0.000044
AFFX-DapX-5_at 0.4 A 0.824672
AFFX-DapX-M_at 2.2 A 0.868639
AFFX-DapX-3_at 3.4 A 0.760937
AFFX-LysX-5_at 0.9 A 0.897835
AFFX-LysX-M_at 10.2 A 0.327079
AFFX-LysX-3_at 1.4 A 0.876448
AFFX-PheX-5_at 1.3 A 0.966335
AFFX-PheX-M_at 0.7 A 0.949771
AFFX-PheX-3_at 12.9 A 0.165861
AFFX-ThrX-5_at 8.2 A 0.175307
AFFX-ThrX-M_at 0.6 A 0.876448

Total number of rows: 45101

Table truncated, full table size 1185 Kbytes.

Supplementary data files not provided

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