Influenza-free, castrated and descented male Fitch ferrets (Mustela putorius furo), 36 to 45 weeks of age, were purchased from Triple F Farms (Sayre, PA). On 0 days post infection (DPI) ferrets in the infection group (n=35) were anesthetized and then received 500 µL of serum free cell medium containing SARS-CoV TOR2 per nare for a total of 1 mL delivered intranasally and a dose of 103 TCID50. Ferrets in the mock-infected group (n=3) were treated in an identical manner but received 1 ml of the vehicle (serum free cell medium) alone intranasally. On 29 DPI, or 0 days post-challenge (DPC), 18 of the original SARS-CoV-infected ferrets were inoculated again (challenged) with 103 TCID50 SARS-CoV TOR2. The three mock-infected ferrets were sacrificed at 2 DPI. Three SARS-CoV-infected ferrets were also sacrificed at 2, 3, 5, 7, 14, and 28 DPI, as well as 2, 3, 5, 7, 14, and 28 DPC (or 31, 32, 34, 36, 43, and 57 DPI). Total RNA was isolated from fresh lung tissue collected during all sacrifice time points using TRIzol reagent by standard protocol (Invitrogen, Carlsbad, CA).
Extracted molecule
total RNA
Extraction protocol
TRIzol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix Two-Cycle protocol from 100 ng total RNA (Expression Analysis Technical Manual, 2004, Affymetrix).
Hybridization protocol
Following fragmentation, 10 microgram of cRNA were hybridized for 16 hr at 45C on GeneChip Canine Genome 2.0 Array in Affymetrix Hybridization Oven 640. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G
Description
Ferret lung with SARS on Day 32 post infection, Day 3 post challenge, biological rep2
Data processing
The data were analyzed with PLIER using ArrayAssist (Stratagene) default analysis settings.
