|
| Status |
Public on Feb 21, 2010 |
| Title |
SR-A-infected CEF, biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
SR-A-infected CEF
|
| Organism |
Gallus gallus |
| Characteristics |
chicken embryonic fibroblast cells derived from day 10 embryos (white leghorn strain)
|
| Treatment protocol |
CEF were infected with the RSV strains wt SR-A, ts NY72-4 and td NY315 or with RCASBP-A, a derivative of avian sarcoma viruses lacking a viral Src gene. CNR cells do not proliferate in the absence of serum or a transforming v-Src kinase. Therefore CNR were infected with the temperature-sensitive mutant NY72-4 RSV at the permissive temperature of 37˚C and were expanded at the permissive temperature of 37˚C. Populations of ts NY72-4 RSV-infected CEF were cultured at the non-permissive temperature of 41.5˚C until transferred to the permissive temperature of 37˚C to induce transformation.
|
| Growth protocol |
CNR and CEF were cultured in high glucose Richter’s modified MEM medium (HyClone) supplemented 5% cosmic calf serum (HyClone), 5% tryptose phosphate broth, glutamine, penicillin and streptomycin. All studies were performed with actively dividing cells cultured in medium replenished the day before sample preparation to avoid starvation and acidosis of the transformed cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cell RNA was isolated using Trizol reagent (Invitrogen) according to manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
15 µg of total RNA was used to prepare biotin labeled complimentary RNA (cRNA). cDNA was generated by one-cycle target labeling protocol using SuperScript II (Invitrogen). Biotinylated cRNA was generated from purified cDNA using the Enzo IVT kit.
|
| |
|
| Hybridization protocol |
cRNA was fragmented and hybridized onto Affymetrix Chicken GeneChip arrays for 16 hours at 45˚C. Washing, staining and scanning was done in accordance with the Affymetrix EukGE-WS2v4 protocol.
|
| Scan protocol |
Scanning was done using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from SR-A-infected CEF
|
| Data processing |
CEL files were analysed using dChip software version 2007. Array data were normalized against the median intensity array for each experiment using the invariant set normalization method. Median array intensities for the experiments were scaled to the same value prior to normalization in order to provide comparable probe intensities for inter-experimental comparison. Expression levels were determined by the model-based expression index method using perfect-match probes only. CNR samples were normalized separately from the CEF samples to prevent skewing from tissue effects.
|
| |
|
| Submission date |
Jan 21, 2009 |
| Last update date |
Jan 14, 2010 |
| Contact name |
Bart Michal Maslikowski |
| E-mail(s) |
[email protected]
|
| Organization name |
McMaster University
|
| Department |
Biology
|
| Lab |
LS-422
|
| Street address |
1280 Main St. West
|
| City |
Hamilton |
| State/province |
Ontario |
| ZIP/Postal code |
L8S 4K1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL3213 |
| Series (1) |
| GSE14489 |
Gene profiling of v-Src transformed primary chicken cells |
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