|
| Status |
Public on Jun 30, 2012 |
| Title |
Patient 4 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood, patient 4
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Peripheral blood
cell type: circulating tumor cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Circulating Tumor Cells (CTC) were immunoisolated from 7.5ml of peripheral blood from six metastatic colorectal patients. Blood was also obtained from three healthy donors. After immunoisolation, RNA was purified with the Qiamp Viral Mini kit (Qiagen) and globally amplified with a whole transcriptome amplification system (WTA2, Sigma-Aldrich). Final cDNA was purified by using the Qiaquick system (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
1.65 µg of cDNA was ULS-Cy3 labeled following manufacturer instructions. Samples were incubated at 85ºC for 30 minutes. After removing non-reacted ULS, labeling degrees were evaluated as recommended. For detailed methods see Agilent G4112-90000 protocol.
|
| |
|
| Hybridization protocol |
Labeled cDNA was mixed with the Agilent hybridization master mix, incubated at 95ºC for 3 minutes. Afterwards, samples were dispensed onto 4x44K microarrays. For detailed methods see Agilent G4112-90000 protocol.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um).
|
| Description |
Gene expression of immunoisolated CTC
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95 and Grid:016528_D_F_20070525) to obtain Processed Signal intensities. The following features and/or genes which did not conform to the established quality criteria were filtered: i) non-uniform pixel distributed outliers and population replicate outliers according to the default Agilent feature extraction criteria; ii) features whose ratio between processed signal and its error was below 2; iii) spots not differentiated from background signal (as estimated for each spot). Normalization was done with the quantile method implemented in the Limma Bioconductor package.
|
| |
|
| Submission date |
Jul 28, 2011 |
| Last update date |
Jun 30, 2012 |
| Contact name |
Miguel Abal |
| E-mail(s) |
miguel.abal.posada@sergas.es
|
| Phone |
0034981955073
|
| Organization name |
University Clinical Hospital
|
| Department |
Medical Oncology
|
| Lab |
Translational Oncology Laboratory
|
| Street address |
Trav Choupana s/n
|
| City |
Santiago de Compostela |
| State/province |
A Coruña |
| ZIP/Postal code |
15706 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE31023 |
Molecular characterisation of circulating tumor cells in human metastatic colorectal cancer |
|
