|
| Status |
Public on Apr 28, 2009 |
| Title |
STAT1 shRNA-transfected STAT1 knockdown SCC61 tumor xenograft, untreated control, technical replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
SCC61 STAT1 knockdown tumor, untreated control
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: STAT1 knockdown
cell line: SCC61
|
| Treatment protocol |
Ionizing radiation (IR) was delivered in 5 Gy fractions over six consecutive days (total 30 Gy) when tumors reached an average size of 160 mm^3 using a Philips RT 250 X-ray generator with a dose rate of 1.65 Gy/min. When tumor volume reached 1,000 mm^3, mice were euthanized by using CO2 followed by cervical dislocation. Tumors were excised, snap-frozen in liquid nitrogen, and stored at -80 °C until RNA extraction.
|
| Growth protocol |
The human squamous cell carcinoma SCC61 cell line was stably transfected with a control vector (SCC61 wild-type [WT]) or one expressing a short hairpin RNA to STAT1 (SCC61 knockdown [KD]) and maintained as previously described (Khodarev NN, et al., 2007). Tumor xenografts were established by subcutaneous injection of 10^7 cells in 100 µL of PBS into the right hind limbs of 6-week-old female athymic mice (FCRI-Taconic).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen tumor xenografts were sectioned into pieces approximately 5 mm^3 in size and soaked overnight in RNAlater-ICE solution (Applied Biosystems-Ambion). Samples were spun, washed in RLT buffer (QIAGEN), and homogenized on ice using a mechanical glass-Teflon homogenizer set at 3,000 rpm. Further purification was performed using TRIzol reagent (Invitrogen Life Sciences) as previously described (Khodarev NN et al., 2002). The quality of samples was assessed using gel electrophoresis in 1.8% agarose and spectrophotometry.
|
| Label |
biotin
|
| Label protocol |
Ribosomal RNA was removed from total RNA extracts using RiboMinus™ Transcriptome Isolation Kit (Invitrogen). Samples were subsequently enzymatically fragmented and biotinylated using GeneChip® Whole Transcript (WT) Sense Target Labeling (Affymetrix).
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| |
|
| Hybridization protocol |
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99 °C for 5 minutes, then 45 °C for 5 minutes centrifuge at max speed for 1 minute • Transfer 200 μl of hybridization solution to each array, then tape holes and parafilm • Hybridize for 17 hours in 45 °C at 60 rpm • Fluidics washing is FS450_0001
|
| Scan protocol |
Affymetrix GeneChip® Scanner 3000 7G using GCOS version 1.4 software with default settings
|
| Description |
STAT1 shRNA-transfected STAT1 knockdown SCC61 tumor xenograft, untreated control, pooled sample from 3 independent tumors normalized to total RNA
|
| Data processing |
Expression Console software version 1.1
|
| |
|
| Submission date |
Apr 27, 2009 |
| Last update date |
Apr 27, 2009 |
| Contact name |
Sean Pravin Pitroda |
| E-mail(s) |
spitroda@uchicago.edu
|
| Organization name |
The University of Chicago
|
| Department |
Radiation and Cellular Oncology
|
| Street address |
5841 South Maryland Avenue, MC 1105
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE15845 |
Knockdown of STAT1 in SCC61 tumor xenografts leads to alterations in the expression of energy metabolic pathways |
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