|
| Status |
Public on Jan 13, 2011 |
| Title |
inVitro6-Hypox_HAg6P |
| Sample type |
RNA |
| |
|
| Source name |
9L glioma cells
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Fisher
cell type: 9L glioma cells
condition: Cells exposed to hypoxia
|
| Treatment protocol |
For in vivo samples: LCM was applied to EF5-positive and negative areas. For in vitro samples: To initiate the experiment, cells were either incubated at ~20% O2 (“Normoxia”) or 0.2% O2 (“Hypoxia”) for 6h or 16h. Cells were subsequently washed with HBSS (Invitrogen), detached with 0.25% trypsin-EDTA and resuspended in RNA lysis solution (Ambion) at a concentration of 50,000cells/100μl lysis solution.
|
| Growth protocol |
Six week old male Fischer 344 rats (F344/Ncr, NCI-Frederick, MD) were inoculated via subcutaneous injection on the hind flank with 9L cells (106) to generate passage ‘zero’ tumors (p0). The tumor was excised, cut into 1-2mm3 pieces and used to generate subsequent passages. The epigastric tumors were initiated by tying p5 or p6 tumor pieces onto the epigastric artery-vein pair. 9L gliosarcoma cells were grown in MEM media with 12% newborn calf serum with twice-weekly transfer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNAqueous-micro kit from Ambion which includes an application for LCM-generated tissue was used to isolate total RNA. A DNase I treatment was performed according to the manufacturer’s protocol. An additional centrifugation step (14,000rpm for 1.5 min) and subsequent supernatant removal were performed to ascertain that all resin had been eliminated. Total RNA was linearly amplified using the NuGEN Ovation WT-Pico kit. Briefly, total RNA (2ng/sample) was reverse transcribed to generate cDNA that incorporated an RNA primer binding sequence. After second-strand synthesis, cDNA templates were amplified by ribo-SPIA. T
|
| Label |
Biotin
|
| Label protocol |
The resulting amplified cDNA was fragmented, biotinylated and hybridized to Rat Gene 1.0ST Arrays (Affymetrix).
|
| |
|
| Hybridization protocol |
Microarrays were stained with streptavadin-phycoerythrin and incubated with biotinylated anti-streptavadin to enhance fluorescence signal detection. Comparison of signal intensity yielded relative gene expression under hypoxia/normoxia.
|
| Scan protocol |
standard Affymetrix scan protocol
|
| Data processing |
Affymetrix .cel files were imported into Partek Genomics Suite (v6.5b, Partek Inc., St. Louis, MO). Robust Multichip Average (RMA) normalization was applied.
|
| |
|
| Submission date |
Dec 09, 2010 |
| Last update date |
Jan 13, 2011 |
| Contact name |
Constantinos Koumenis |
| E-mail(s) |
koumenis@xrt.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Radiation Oncology
|
| Street address |
3620 Hamilton Walk
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19146 |
| Country |
USA |
| |
|
| Platform ID |
GPL6247 |
| Series (1) |
| GSE25980 |
In vitro and in vivo analysis of hypoxic gene expression in rat gliomas |
|
