Patient: sporadic unilateral Rb, female, age at dx in days:985, primary enucleation, original RNA ID M19485 LOH 16q: yes
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).
