|
| Status |
Public on Sep 01, 2011 |
| Title |
SARS-CoV-HC/SZ/6103-day-21-replicate-2 |
| Sample type |
RNA |
| |
|
| Source name |
SARS-CoV-HC/SZ/6103-day-21-replicate-2
|
| Organism |
Macaca mulatta |
| Characteristics |
infection: SARS-CoV-HC/SZ/6103
time: day 21
tissue: lung
|
| Treatment protocol |
Animals were infected with either Urbani GZ02 or HC/SZ/61/03 under anesthesia through a combination of intratracheal (4 ml) and intranasal (0.5 ml per nostril) installation with a suspension containing 2x106 plaque forming units (pfu) per ml DMEM (total infectious dose = 1x107 pfu) of each of the 3 different SARS-CoV strains. Three animals were mock infected with DMEM only. Animals were anesthetized for challenge clinical examination temperature respiration rate chest radiographs blood draw and swabs of nasal oral and rectal mucosa on days 01247 11 and 14 p.i. Three animals from each infected group and 1 control animal were euthanized and necropsied on days 1 4 and 14 p.i. with collection of clinical specimens. Experiments were conducted under BSL-3+ conditions and approval for animal experiments was obtained from the institutional Animal Care and Use Committee by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved facility
|
| Growth protocol |
All viruses were propagated on Vero E6 cells in Dulbecco's minimal essential medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum L-Glutamine penicillin (10000 IU/mL) and streptomycin (10000 IU/mL) at 37°C in a humidified CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue samples were placed in RNAlater (Qiagen) for 11 days at 4°C. RNAlater was removed and samples were homogenized in 1 ml of Trizol for subsequent RNA extraction. Upon thawing 200 µl chloroform was added vials were vortexed and centrifuged at 16000 x g for 15 min. RNA containing aqueous phase of 350-550 µl was collected from each sample and passed through Qiashredder column (Qiagen) at 21000 x g for 2 minutes to homogenize any remaining genomic DNA in the aqueous phase. RNA was purified using RNeasy 96 kit (Qiagen) as described previously except RNA samples were treated with DNase I during extraction
|
| Label |
biotin
|
| Label protocol |
Five nanograms of RNA sample from tissue with equivalent viral RNA levels was processed according to manufacturer's instructions using WT-Ovation™ Pico system RNA Amplification System (Nugen Inc.). Each sample was fragmented and labeled according to manufacturer's instructions for standard format anti-sense (AT) GeneChip arrays (Nugen Inc).
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| |
|
| Hybridization protocol |
Samples were hybridized onto Affymetrix GeneChip Rhesus Macaque Genome Arrays (Affymetrix) according to manufacturer's recommendations
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.
|
| Description |
Gene expression data from__SARS-CoV-HC/SZ/6103-day-21-replicate-2
|
| Data processing |
The data were analyzed with GCOS 1.4 using Affymetrix default analysis with a scale filter for Se genes setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
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| |
|
| Submission date |
Sep 02, 2010 |
| Last update date |
Sep 01, 2011 |
| Contact name |
Dan Sturdevant |
| E-mail(s) |
[email protected]
|
| Phone |
4063639248
|
| Organization name |
NIH
|
| Department |
NIAID
|
| Lab |
RTS
|
| Street address |
903 S 4th street
|
| City |
Hamilton |
| State/province |
MT |
| ZIP/Postal code |
59840 |
| Country |
USA |
| |
|
| Platform ID |
GPL3535 |
| Series (1) |
| GSE23955 |
Comparative Pathogenesis of Three Human and Zoonotic SARS-CoV Strains in Cynomolgus Macaques |
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