|
| Status |
Public on Sep 26, 2012 |
| Title |
pig 1421 at 14 day vs. at 0 day 1421_14_0 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
1421_14
|
| Organism |
Sus scrofa |
| Characteristics |
animal id: 1421
response: high viral load + high weight gain
infection: Porcine Reproductive and Respiratory Syndrome Virus
time (d): 14
tissue: blood
group_dpi: hvhg_14
breed: commercial crossbred pigs
|
| Treatment protocol |
After a 7-day acclimation period and antibiotic treatments, pigs were intramuscularly and intranasally infected with a known isolate of the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) (105 tissue culture infectious dose50 of NVSL 97-7985).Blood samples were collected in Tempus™ Blood RNA Tubes (Life Technologies, Carlsbad, CA) at several time-points post-infection, including 0, 4, 7, 11, 14, 21, 28 and 42 days post-infection (DPI). Individual animal weight was measured at weekly intervals. Serum viral level was quantified using a semi-quantitative TaqMan PCR assay.
|
| Growth protocol |
Crossbred commercial pigs (~200) from PPRS Host Genetics Consortium (PHGC) trial one were transported to a Kansas State University bio-secure testing facility at weaning (11 to 21 d. old) and allocated to pens (10 to 15 pigs/pen). Pigs came from PRRSV-, Influenza virus- and Mycoplasma hyopneumoniae-free farms.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tempus tube collected blood samples of PHGC pigs were stored at -20 until RNA was extracted
|
| Label |
Cy3
|
| Label protocol |
One µg of total RNA was reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Following first-strand and second-strand synthesis and purification, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs. After purification, 10 ug of aRNA was labeled with N-hydroxysuccinate (NHS) ester Cy3 or Cy5 (GE Healthcare, UK).
|
| |
|
| Channel 2 |
| Source name |
1421_0
|
| Organism |
Sus scrofa |
| Characteristics |
response: high viral load + high weight gain
animal id: 1421
infection: Porcine Reproductive and Respiratory Syndrome Virus
time (d): 0
tissue: blood
group_dpi: hvhg_0
breed: commercial crossbred pigs
|
| Treatment protocol |
After a 7-day acclimation period and antibiotic treatments, pigs were intramuscularly and intranasally infected with a known isolate of the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) (105 tissue culture infectious dose50 of NVSL 97-7985).Blood samples were collected in Tempus™ Blood RNA Tubes (Life Technologies, Carlsbad, CA) at several time-points post-infection, including 0, 4, 7, 11, 14, 21, 28 and 42 days post-infection (DPI). Individual animal weight was measured at weekly intervals. Serum viral level was quantified using a semi-quantitative TaqMan PCR assay.
|
| Growth protocol |
Crossbred commercial pigs (~200) from PPRS Host Genetics Consortium (PHGC) trial one were transported to a Kansas State University bio-secure testing facility at weaning (11 to 21 d. old) and allocated to pens (10 to 15 pigs/pen). Pigs came from PRRSV-, Influenza virus- and Mycoplasma hyopneumoniae-free farms.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tempus tube collected blood samples of PHGC pigs were stored at -20 until RNA was extracted
|
| Label |
Cy5
|
| Label protocol |
One µg of total RNA was reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Following first-strand and second-strand synthesis and purification, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs. After purification, 10 ug of aRNA was labeled with N-hydroxysuccinate (NHS) ester Cy3 or Cy5 (GE Healthcare, UK).
|
| |
|
| |
| Hybridization protocol |
Labeled aRNAs were purified and combined with 70 µl of Slide Hyb #1 solution (Ambion, Inc.) and denatured at 70C for 3 min. Hybridizations were performed in sealed hybridization cassettes (ArrayIt, TeleChem International, Inc., Sunnyvale, CA, USA) for 18 h at a humid 54C. Following hybridization, slides were washed in 2X SSC/0.5% SDS and 0.1X SSC/0.1% SDS solutions for 10 min each. The slides were rinsed in a 0.1X SSC solution and nuclease-free water and dried by centrifugation.
|
| Scan protocol |
Fluorescent images were detected by a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA) and fluorescence intensity data were collected using GenePix software (Molecular Devices) after spot alignment. Median intensity values for each dye channel were stored as comma-separated values data files.
|
| Description |
BLOOD EXPRESSION PRRSV infection title indicates pig ID and days post infection
|
| Data processing |
Median intensities were extracted and normalized using a within print-tip loess location normalization implemented in NormalizeWithinArrays() in LIMMA. Background was corrected following Ritchie et al. (2007) method using normexp() function of LIMMA with offset=50. The resulting normalized ratios were expressed in the log2 scale.
|
| |
|
| Submission date |
Sep 25, 2012 |
| Last update date |
Oct 03, 2012 |
| Contact name |
juan p steibel |
| E-mail(s) |
[email protected]
|
| Organization name |
Michigan State University
|
| Street address |
1205 I Anthony Hall
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48842 |
| Country |
USA |
| |
|
| Platform ID |
GPL7435 |
| Series (1) |
| GSE41144 |
Whole blood response of Pigs to infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) |
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