|
| Status |
Public on Jan 15, 2011 |
| Title |
BLN_5089_VS_BLN_5091 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BLN_5089
|
| Organism |
Sus scrofa |
| Characteristics |
sample type: MNW2B_3dpi
infection: MNW2B
time: 3 dpi
tissue: tracheobronchial lymph nodes
|
| Treatment protocol |
Pigs were either infected intranasally and intramuscularly with heterologous Acute-type Minnesota strain (MNW2B) PRRSV (n=7) or NC Powell (n=4), or vaccinated with contemporary Modified Live Virus (MLV) PRRS ATP vaccine (Ingelvac®) (n=3) at a dose of 104.5 TCID50. The control group consisted of naïve pigs (n=3).
|
| Growth protocol |
4-week old pigs (medicated and early-weaned ) were used for the study.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pigs were sacrificed at 3-7 days post infection (dpi). The 3 vaccinated pigs were sacrificed at 4dpi. Naïve pigs were sacrificed the following week. Tissues of tonsil, tracheobronchial lymph nodes (TBLN), Cranial lung (CR Lung), and distal lung (D Lung) were collected and placed at -80°C (NC Powell infected lung tissues were not available). RNA isolation and purification was performed using TRIzol® reagent and PureLinkTM kit (Invitrogen). RNA concentration and quality was determined with an RNA 6000 Pico LabChip kit using an Agilent 2100 Bioanalyzer (Agilent Technologies,Inc.)
|
| Label |
Alexa 555
|
| Label protocol |
For each sample, 1 μg of total RNA was reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion Inc.) 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 aRNA purification,some aRNAs were used for the RNA/aRNA comparison; 10 μg of aRNAs were labelled with Alexa Fluor® 555 and Alexa Fluor® 647 dyes (Invitrogen) as appropriate for the experimental design. The labeled aRNAs (2.5 μg) were purified and combined with 65μl of Slide Hyb #1 solution (Ambion Inc.) and denatured at 70 ℃ for 5 min.
|
| |
|
| Channel 2 |
| Source name |
BLN_5091
|
| Organism |
Sus scrofa |
| Characteristics |
sample type: MNW2B_5dpi
infection: MNW2B
time: 5 dpi
tissue: tracheobronchial lymph nodes
|
| Treatment protocol |
Pigs were either infected intranasally and intramuscularly with heterologous Acute-type Minnesota strain (MNW2B) PRRSV (n=7) or NC Powell (n=4), or vaccinated with contemporary Modified Live Virus (MLV) PRRS ATP vaccine (Ingelvac®) (n=3) at a dose of 104.5 TCID50. The control group consisted of naïve pigs (n=3).
|
| Growth protocol |
4-week old pigs (medicated and early-weaned ) were used for the study.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pigs were sacrificed at 3-7 days post infection (dpi). The 3 vaccinated pigs were sacrificed at 4dpi. Naïve pigs were sacrificed the following week. Tissues of tonsil, tracheobronchial lymph nodes (TBLN), Cranial lung (CR Lung), and distal lung (D Lung) were collected and placed at -80°C (NC Powell infected lung tissues were not available). RNA isolation and purification was performed using TRIzol® reagent and PureLinkTM kit (Invitrogen). RNA concentration and quality was determined with an RNA 6000 Pico LabChip kit using an Agilent 2100 Bioanalyzer (Agilent Technologies,Inc.)
|
| Label |
Alexa 647
|
| Label protocol |
For each sample, 1 μg of total RNA was reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion Inc.) 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 aRNA purification,some aRNAs were used for the RNA/aRNA comparison; 10 μg of aRNAs were labelled with Alexa Fluor® 555 and Alexa Fluor® 647 dyes (Invitrogen) as appropriate for the experimental design. The labeled aRNAs (2.5 μg) were purified and combined with 65μl of Slide Hyb #1 solution (Ambion Inc.) and denatured at 70 ℃ for 5 min.
|
| |
|
| |
| Hybridization protocol |
Pigoligoarray hybridizations were performed in sealed hybridization cassettes (ArrayIt, TeleChem International, Inc.) for 18 h at a humid 54 ℃. A low stringency experiment was also conducted at MSU with hybridizations at 42 ℃. Following hybridization, slides were washed in 2×SSC/0.5% SDS and 0.1× SSC/0.1% SDS solutions for 10 min each. The slides were rinsed in a 0.1×SSC solution and nuclease-free water and dried by centrifugation.
|
| Scan protocol |
Fluorescent images were detected by an Axon GenePix® 4000B scanner (Molecular Devices), and fluorescence intensity data were collected using GENEPIX® PRO 6 software (Molecular Devices) after spot alignment.
|
| Description |
BLN EXPRESSION PRRSV infection title indicates Treatment and days post infection
|
| Data processing |
Median intensities were extracted and normalized using a within print-tip lowess location normalization and an overall scale normalization. The resulting normalized data were expressed in the log2 scale.
|
| |
|
| Submission date |
Jan 14, 2011 |
| Last update date |
Jan 15, 2011 |
| 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) |
| GSE26642 |
Pathology and Protective Immune Response in Pigs Infected with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) of High and Low Virulence |
|
