|
| Status |
Public on Jun 07, 2012 |
| Title |
HumanTumor_FreshFrozen_CRC103 |
| Sample type |
RNA |
| |
|
| Source name |
human tumor, colorectal primary, fresh frozen tissue
|
| Organism |
Homo sapiens |
| Characteristics |
primary tumor site: colorectal
sample type: patient tumor
metastatic tumor site: liver
|
| Treatment protocol |
N/A
|
| Growth protocol |
Human tumors were resected at time of surgery and flash frozen in OCT (Optimal Cutting Temperature) on dry ice. To generate patient-derived colorectal explants (PDCCEs), the collected human tumor samples were washed with PBS and then minced into 2~3 mm cubes. The samples were then placed in an enzyme medium (RPMI media containing collagenase IV, hyaluronidase and deoxyribonuclease) and agitated at room temperature for 18-24h. After agitation, the cells were spun down, washed with PBS and passed through a cell strainer. To eliminate red blood cells, 2-3 ml of red blood cell lysis buffer were added to the cells and incubated at 37°C for 5 min. After washing with PBS, the cells were centrifuged at 2000 rpm for 15 min at room temperature, resuspended in serum free-RPMI Matrigel mixture (1:1 volume) and then injected into the flanks of 4-week-old female JAX NOD.CB17-PrkdcSCID-J mice under a Duke IACUC approved protocol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Human tumors were flash frozen in OCT at time of surgical resection. PDCCE tumors were harvested at the end of 3 weeks and flash frozen in OCT. For both human tumors and PDCCEs, an initial section was stained with Hematoxylin and Eosin for histological characterization of the tissue, and the sample was subsequently macrodissected to ensure > 80% tumor. Approximately 100 ug of tissue was macrodissected, and total RNA was isolated from the homogenized tissue using the RNAase Isolation Kit (Qiagen, Valencia, Ca). Additionally, PDCCEs were also fixed in formalin overnight or 3 days and paraffin embedded the following day. RNA from the FFPE tumor samples was then extracted using the High Pure RNA Paraffin Kit (Roche).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 3-4 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
The resulting biotin-labeled RNA samples were then hybridized with Affymetrix U133A 2.0 GeneChip arrays according to the manufacturer's instructions (Affymetrix, Santa Clara, CA).
|
| Scan protocol |
GeneChips were scanned using the Gene Array Scanner (Affymetrix) following the manufacturer’s instructions (Affymetrix, Santa Clara, CA).
|
| Data processing |
The data were analyzed using the R statistical platform with the Bioconductor affymetrix package.
|
| |
|
| Submission date |
Jan 17, 2012 |
| Last update date |
Jun 07, 2012 |
| Contact name |
David Hsu |
| Organization name |
Duke University
|
| Department |
Medical Oncology
|
| Street address |
905 S. LaSalle St. Rm 3008
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE35144 |
Molecular Evaluation of Patient-Derived Colorectal Cancer Explants as a Pre-clinical Mouse Model of Colorectal Cancer |
|
