|
| Status |
Public on Mar 23, 2015 |
| Title |
leukocytes dog 4-stimulated-biological replicate 4 |
| Sample type |
RNA |
| |
|
| Source name |
Canine leukocytes, stimulated
|
| Organism |
Canis lupus familiaris |
| Characteristics |
cell type: T-cells
gender: female
age (years): 1
breed: Bullmastiff
treament: T-cells stimulated with PHA
|
| Treatment protocol |
Blood was collected into EDTA coated vacutainers, and the leukocyte fraction was isolated by gradient centrifugation. Cells were divided into two aliquots and each were resuspended in 1 ml Dulbecco’s Modified Eagle Medium media. Both were incubated for 4 hours, one in the presence of 5μg/ml of PHA, a mitogen for T-cell activation, the other served as non-stimulated control cells. After incubation the cells were collected by centrifugation, and then lysed in RLT buffer (QIAmp; Qiagen Aust). Once lysed samples were immediately frozen until further use (within 48hr).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from samples using a modified extraction procedure and micro-spin columns (QIAmp; Qiagen, Melbourne, Vic). The lysate was pipetted directly into a QIAshredder spin column following incubation in a 37˚C water bath until completely thawed and salts dissolved. 70% ethanol was added to the homogenized lysate before it was transferred into a QIAamp spin column and washed using a high-salt buffering system allowing RNA to bind to the QIAmp membrane as contaminants are removed. Purified RNA was eluted in 30 μl of RNase-free water followed by a second elution in 50 μl and stored at -80˚C.
|
| Label |
biotin
|
| Label protocol |
Labelled and hybridized to an Affymetrix® Canine Gene 1.0 ST Array according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Labelled and hybridized to an Affymetrix® Canine Gene 1.0 ST Array according to the manufacturer’s instructions.
|
| Scan protocol |
Genechips were scanned using GeneChip Scanner 3000 (Affymetrix). Probe intensities were obtained using GCOS operating software (Affymetrix)
|
| Data processing |
RMA
|
| |
|
| Submission date |
Jan 21, 2015 |
| Last update date |
Mar 23, 2015 |
| Contact name |
Peter Williamson |
| Organization name |
University of Sydney
|
| Department |
Faculty of Veterinary Science
|
| Street address |
Rm 552 RMC Gunn Building B19
|
| City |
The University of Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19674 |
| Series (1) |
| GSE65158 |
T-cell activation and early gene response in dogs |
|
