|
| Status |
Public on Nov 01, 2011 |
| Title |
SM001 SARS 10^4pfu D2 2 |
| Sample type |
RNA |
| |
|
| Source name |
SM001 SARS 10^4pfu D2 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: Female
time (post-infection): 2 day
age: 20 week old
treatment: SARS CoV MA15 infected with 10^4 PFU
tissue: lung
|
| Treatment protocol |
Specific lobs of the lung from each animal were harvested and briefly rinse tissue in cold (4ºC) PBS. Following the RNAlater (Ambion) protocol, tissue was cut into small chunks (<0.5cm in any single dimension) and place immediately into a 10-20 volumes (w/v) (e.g. 100mg/ml) RNAlater. After a 4ºC incubation for overnight, samples were stored at -80ºC further processing. Lung tissue was removed from RNAlater, washed in a small volume of Trizol, homogenized in 10-20 volumes (w/v) TRIzol and stored at -80°C until RNA isolation.
|
| Growth protocol |
Twenty-week-old C57BL/6 mice were infected by intranasal instillation of 10^2,10^3, 10^4 or 10^5 PFU of SARS CoV MA15 in 50 µl of PBS or mock-infected with PBS alone. At days 1, 2, 4 and 7 days post-infection, lungs were harvested.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All TRIzol lysates were processed simultaneously: they were phase-separated, and RNA was isolated from the aqueous phase (diluted 2 fold with RLT buffer) using Qiagen RNeasy Mini columns and the manufacturer’s recommended protocol (Qiagen Inc., Valencia, CA). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the nanochip format, and only intact RNA was used for microarray analyses.
|
| Label |
Cy3
|
| Label protocol |
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for the Cy3-cDNA probe preparation.
|
| |
|
| Hybridization protocol |
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for hybridization and array washing. Two hundred fifty ng of each RNA sample was hybridized to one Agilent 4X44K human HG (Design ID 014850) array.
|
| Scan protocol |
Dry slides were scanned on an Agilent DNA microarray scanner (Model G2505B) using the XDR setting.
|
| Data processing |
Raw images were analyzed using the Agilent Feature Extraction software (version 9.5.3.1) and the GE1-v5_95_Feb07 extraction protocol. All arrays were required to pass Agilent QC flags. Extracted raw data were background corrected using the norm-exp method and quantile normalized using Agi4x44PreProcess and RMA Bioconductor packages.
|
| |
|
| Submission date |
Oct 26, 2011 |
| Last update date |
Nov 01, 2011 |
| Contact name |
Michael Katze |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
Microbiology
|
| Lab |
Michael G. Katze, Ph.D
|
| Street address |
Rosen Building 960 Republican St.
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109-4325 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE33266 |
SM001: SARS CoV MA15 infection of C57Bl/6 mouse model – Data from 4 viral doses at 1, 2, 4 and 7 days post infection. |
|
