|
| Status |
Public on Feb 28, 2010 |
| Title |
EPIC case #4: bronchial carcinoma |
| Sample type |
RNA |
| |
|
| Source name |
peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
bronchial carcinoma
|
| Extracted molecule |
total RNA |
| Extraction protocol |
weWe used snap frozen PBMC enriched blood (~ 300 µl) from the EPIC study for RNA extraction. Blood samples were directly thawed in 5ml of TRI Reagent BD (Molecular Research Center, Inc, USA). RNA was extracted according to the manufactures instruction and stored in 70% ethanol until finalization of preparation and dilution in H2O. For further RNA purification we applied Qiagen MinElute columns (Qiagen, Germany). By this method we extracted 960ng RNA (mean; range 120 – 2500ng). One sample contained no RNA. The OD ratio 260/280 was 2.01 in mean (range 1.76-2.06). For the LC-group, we drew 2.5 ml blood directly into PAXgene vials providing stabilization of the gene expression profile. Samples were rested over night at room temperature and then stored at -80°C until further preparation. RNA was extracted according to the manufactures instruction (Qiagen, Hilden, Germany).
|
| Label |
biotin
|
| Label protocol |
Biotin labeled cRNA preparation was performed using the Ambion® Illumina RNA amplification kit (Ambion, UK). Quality was controlled using a PCR based system1.
|
| |
|
| Hybridization protocol |
For hybridization samples were applied to the arrays and assembled into HybCartridges (Illumina). BeadChips were hybridized for 16h at 55°C on the BeadChip Hyb Wheel. 1.5 μg biotin labeled cRNA was hybridized to Sentrix® whole genome bead chips 6 x 2 V1 and V2 (Illumina, USA) according to the manufacturers instruction. Arrays were washed according to Illumina Wash BeadChip 6 x 2 protocol. Signals were developed with streptavidin Cy3.
|
| Scan protocol |
Scanning was performed on the Illumina(r) BeadStation 500x.
|
| Description |
All samples were taken from smokers. EPIC cases were taken from the EPIC trial (a large prospective epidemiological trial in Europe, and are incident cases), Cosmos and BC cases are prevalent cases from a Cologne, Germany based epidemiological trial.
|
| Data processing |
BeadStudio raw data were normalized using R
|
| |
|
| Submission date |
Sep 13, 2008 |
| Last update date |
Oct 23, 2009 |
| Contact name |
Thomas Zander |
| Organization name |
University Hospital Cologne
|
| Street address |
Kerpenerstr. 62
|
| City |
Cologne |
| ZIP/Postal code |
50924 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6102 |
| Series (1) |
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