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Sample GSM118320 Query DataSets for GSM118320
Status Public on Jan 29, 2007
Title Retinoblatoma M20517
Sample type RNA
Source name retinoblastoma
Organism Homo sapiens
Characteristics Patient: sporadic bilateral Rb, male, age at dx in days:240, primary enucleation, original RNA ID M20519
LOH 16q: no
Treatment protocol no treatment
Growth protocol no in vitro culture
Extracted molecule total RNA
Extraction protocol Isolation of total RNA was performed by a modified Qiagen RNeasy Mini Protocol for the isolation of totel RNA from animal tissues (QIAgen, Hilden, Germany). All the buffers and columns that are contained in this kit were used, except when specified. A 30 mg tumour sample is placed in 800 µl Buffer RLT (ß-ME 10µl/ml, Sigma-Aldrich, Taufkirchen, Germany) in a Lysing Matrix D column (Q.BIOgene, Heidelberg, Germany) and grinded in the Lysing Matrix for 45 s at 6.5 (Fast Prep, FP 120, Bio 101 Systems, Q.BIOgene). Dry ice is used to cool down the sample after grinding. At room temperature, the samples are shaken, vortexed and spun down for 5 min at 13,000 rpm at 4°C. The supernatant is taken out and placed in a new 1.5 ml tube, and spun down again in the same conditions as before. Leaving the protein pellet intact, the supernatant is placed in a 1.5 ml tube to which 750 µl Ethanol 70% is added, and then vortexed. The 1.5 ml solution obtained in this step is then placed on an RNeasy mini spin column and centrifuged for 15 s at 10,000 rpm at 4°C (in 2 steps). The flow-through is discarded. Digestion with DNaseI (QIAgen) is used to avoid the presence of DNA in the final extract. For this, 350 µl Buffer RW1 is added to the spin column and centrifuged for 15 s at 10,000 rpm. The flow-through is discarded. A mix of 10 µl DNaseI stock solution and 70 µl buffer RDD is thoroughly pipetted on the membrane of the column and incubated for 15 min at room temperature. After adding 350 µl buffer RW1 the column is centrifuged for 15 s at 10,000 rpm. The column is then placed on a new collection tube, 500 µl buffer RPE (with Ethanol) is added and spun down for 2 min at 13,000 rpm. The column placed on a new collection tube and the RNA is eluted with 50 µl RNase free water by centrifugation for 1 min at 10,000 rpm. The elution step is repeated one time..
Label biotin
Label protocol Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2.5 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at 45C on HG-U133A Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Description Aim: to compare the transcriptomes of retinoblastoma with LOH on 16q to tumors without alterations on 16q.
Data processing The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1000. Differentially expressed target genes were identified using Significance Analysis of Microarrays (SAM).
Submission date Jul 03, 2006
Last update date Jan 29, 2007
Contact name Ludger Klein-Hitpass
Phone +49 201 723 85552
Organization name Institut fuer Zellbiologie
Department Universitaetsklinikum
Lab BioChip Lab
Street address Virchowstr. 173
City Essen
ZIP/Postal code D-45122
Country Germany
Platform ID GPL96
Series (1)
GSE5222 Expression data from retinoblastoma

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 1019.7 P 0.001102
AFFX-BioB-M_at 2143.9 P 0.000127
AFFX-BioB-3_at 1602.9 P 0.00011
AFFX-BioC-5_at 2653.7 P 0.00007
AFFX-BioC-3_at 2037.2 P 0.000044
AFFX-BioDn-5_at 4639 P 0.000052
AFFX-BioDn-3_at 17191.7 P 0.000044
AFFX-CreX-5_at 27632.2 P 0.000044
AFFX-CreX-3_at 42939.3 P 0.000044
AFFX-DapX-5_at 2693.4 P 0.00007
AFFX-DapX-M_at 6525.5 P 0.000127
AFFX-DapX-3_at 9956.2 P 0.000044
AFFX-LysX-5_at 16480 P 0.000044
AFFX-LysX-M_at 27365.4 P 0.000044
AFFX-LysX-3_at 33337.4 P 0.000044
AFFX-PheX-5_at 53671.7 P 0.000044
AFFX-PheX-M_at 65811.7 P 0.000044
AFFX-PheX-3_at 72285.8 P 0.000044
AFFX-ThrX-5_at 24899.4 P 0.000044
AFFX-ThrX-M_at 24095 P 0.000044

Total number of rows: 22283

Table truncated, full table size 601 Kbytes.

Supplementary data files not provided

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