|
| Status |
Public on Dec 21, 2009 |
| Title |
B6 control 2 |
| Sample type |
RNA |
| |
|
| Source name |
C57BL6/J strain lung RNA, control
|
| Organism |
Mus musculus |
| Characteristics |
tissue: lung
strain: C57BL6/J
age: 15-26 weeks
treatment: untreated
|
| Treatment protocol |
Mice were treated at 8 weeks of age. Lung damage was elicited by whole thorax radiation exposure (18 Gy) using a Gamma cell Cesium-137 unit. The rest of the body was shielded with 3 cm of lead, to reduce the beam strength to 3% in this area. The mice were weighed weekly from eight weeks after radiation and sacrificed when moribund which was defined as loss of > 20% body weight, exhibition of distress through ruffled fur, accelerated breathing and hunched posture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following sacrifice, the right lung was immediately homogenized in 2 mL of Trizol reagent and placed in dry ice. The homogenates were stored at -85 degrees C until RNA isolation. Total lung RNA was extracted according to the manufacturer’s (Sigma) instructions. To define the set of expressed genes the RNA from three post treatment moribund mice, and three samples from control mice, was evaluated for each strain. RNA quality was assessed and confirmed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, California).
|
| Label |
biotin
|
| Label protocol |
After second-strand synthesis, the probe cDNA was purified by phenol chloroform extraction with Phase-Lock tubes (Eppendorf) and redissolved in 20 ml DEPC water. Biotinylated probe was prepared from the entire cDNA reaction mixture with the ENZO bioarray high-yield RNA transcript labeling kit (ENZO diagnostics). The probe synthesis reaction was performed at 378C for 5 hours with occasional agitation. The biotinylated probe was purified by using an RNAeasy spin column (Qiagen), eluted in 80 ml of DEPC water, quantified by spectrophotometry, and analyzed on a nondenaturing gel. The average probe length was reduced by incubating the purified probe in 13 fragmentation buffer for 35 minutes at 958C.
|
| |
|
| Hybridization protocol |
The hybridization mixture was prepared by mixing 15 mg biotinylated probe with control oligonucleotide B2 (final concentration 50 pM; Affymetrix), herring sperm DNA (final concentration 0.1 mg/ml; Research Genetics), acetylated bovine serum albumin (final concentration 0.5 mg/ml; Gibco BRL Life Technologies) in a final volume of 300 ml of 13 MES hybridization buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20; all reagents from Sigma). The hybridization mixture was denatured for 10 minutes at 99C, incubated for 5 minutes at 45C, and spun for 5 minutes in a benchtop microcentrifuge. The microarray was warmed to room temperature and prehybridized in 13 hybridization buffer for 10-20 minutes at 45C. The prehybridization solution was removed and 150 ml of the hybridization mix was added to the array. The array and probe fragments were incubated at 458C overnight (16-20 hours).
|
| Scan protocol |
GeneChips were scanned using a GeneArray Scanner (Agilent technologies)
|
| Description |
Gene expression data from untreated C57BL6/J strain
|
| Data processing |
data was analyzed using the MAS5.0 algorithm.
|
| |
|
| Submission date |
Dec 21, 2009 |
| Last update date |
Dec 21, 2009 |
| Contact name |
Christina Kathleen Haston |
| E-mail(s) |
christina.haston@mcgill.ca
|
| Phone |
514-398-3864 089714
|
| Organization name |
McGill University
|
| Department |
Medicine
|
| Street address |
3626 St. Urbain
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H2X 2P2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE2250 |
Effects of irradiation on mouse lung |
|
