|
| Status |
Public on Sep 08, 2009 |
| Title |
13536745_33_22 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver, Infected
|
| Organism |
Sus scrofa |
| Characteristics |
Pig no. 33, inoculated, no. 8 of 10 replicates, infected in 14-18h with Actinobacillus pleuropneumonia 5b (strain L20).
|
| Treatment protocol |
10 Pigs were inoculated with a total of 1ml McFarland suspension mixed 1:1 with Brain Heart infusion Broth + 0.5% NAD, containing approxemately 0.7 x 10E7 in both nostrils during inhalation, 5 pigs were not inoculated and used as controls, 14-18h post inoculation pigs were sacrificed, tissue samples of approximately 500mg were taken and snap frosen in liquid nitrogen and stored at -80 degrees until RNA extraction
|
| Growth protocol |
15 pigs were used in this study, these were 8-10 weeks old castrates of the race Danish Landrace/Yorkshire/Duroc, from a specific pathogen free (SPF) herd, free of Actinobacillus pleuropneumoniae.
All animal procedures were approved by the Danish Animal Experiments Inspectorate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen RNeasy midi kit according to manufactures protecol with additional DNase treatment (RNase-free DNase, Qiagen) according to manufactures protecol. Quantity of the extracted total RNA was measured using Nanodrop ND-1000 and quality was measured using Agilent 2100 Bioanalyzer
|
| Label |
Oyster 550
|
| Label protocol |
7ug of RNA for each sample was labeled using Genisphere 3DNA Array900 labeling kit according to manufactures protecol for large-scale cDNA synthesis
|
| |
|
| Channel 2 |
| Source name |
Liver, Control
|
| Organism |
Sus scrofa |
| Characteristics |
Pig no. 22, control, no. 3 of 5 replicates, not inoculated.
|
| Treatment protocol |
10 Pigs were inoculated with a total of 1ml McFarland suspension mixed 1:1 with Brain Heart infusion Broth + 0.5% NAD, containing approxemately 0.7 x 10E7 in both nostrils during inhalation, 5 pigs were not inoculated and used as controls, 14-18h post inoculation pigs were sacrificed, tissue samples of approximately 500mg were taken and snap frosen in liquid nitrogen and stored at -80 degrees until RNA extraction
|
| Growth protocol |
15 pigs were used in this study, these were 8-10 weeks old castrates of the race Danish Landrace/Yorkshire/Duroc, from a specific pathogen free (SPF) herd, free of Actinobacillus pleuropneumoniae.
All animal procedures were approved by the Danish Animal Experiments Inspectorate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen RNeasy midi kit according to manufactures protecol with additional DNase treatment (RNase-free DNase, Qiagen) according to manufactures protecol. Quantity of the extracted total RNA was measured using Nanodrop ND-1000 and quality was measured using Agilent 2100 Bioanalyzer
|
| Label |
Oyster 650
|
| Label protocol |
7ug of RNA for each sample was labeled using Genisphere 3DNA Array900 labeling kit according to manufactures protecol for large-scale cDNA synthesis
|
| |
|
| |
| Hybridization protocol |
1. cDNA from two samples (2 x 25ul), 1ul salmon sperm DNA (10ug/ul) and 51ul 2xFormamide-Based Hybridization Buffer (Genisphere) were mixed. 70ul of this solution were applied under a 25x60mm LifterSlip (Erie Scientific).
2. Slides were incubated over-night in a Corning hybridization chamber at 44 degree Celcius in water bath
3. Slides were washed according to manufactures protecol with Wash buffer 1 pre-heated to 44 degree celcius, slides were dryed by centrifugation
4. 3DNA hybridization mix was prepared: 2.5ul of each capture reagent, Oyster 550 and Oyster 650 (3DNA Array900, Genisphere), 41ul SDS-based hybridization buffer (Genisphere), 35ul milliQ water and 1ul salmon sperm DNA (10ug/ul) were mixed.
5. 70ul 3DNA hybridization mix was applied to each slide and incubated in the dark for 4 hours at 62 degree in a water bath
6. Slides were washed according to manufactures protecol with Wash buffer 1 pre-heated to 64 degree celcius, slides were dryed by centrifugation
|
| Scan protocol |
Slides were scanned on a CCD-based ArrayWoRxe auto (Applied Precision)
|
| Description |
13536745_33_22
|
| Data processing |
1. Image processing and spot finding were performed using GenePixPro 6.0 (Molecular Devices Corp.)
2. Low intensity spots were flagged if they did not have at least 45% of their feature pixels more than 2 SD above background. Incorrectly placed spots were flagged manually.
3. Ratio based normalization using default options was performed in Acuity 4.0 (Molecular Devices Corp.)
4. Normalized background substracted medians for all spots were exported to Excel and used to calculate mean ln expression values for each gene in each pig.
5. Mean overall ln expression values was calculated for each gene across the 5 control pigs.
6. These values was respectively subtracted from the mean expression value for each gene in each of the infected pigs, and, there was finally calculated an overall mean ln difference across the 10 infected pigs as opposed to the control pigs.
|
| |
|
| Submission date |
May 09, 2008 |
| Last update date |
Sep 08, 2009 |
| Contact name |
Kerstin Skovgaard |
| E-mail(s) |
[email protected]
|
| Phone |
+4572346362
|
| Organization name |
National Veterinary Institute
|
| Department |
Immunology and Parasitology
|
| Street address |
Bülowsvej 27
|
| City |
Copenhagen |
| ZIP/Postal code |
1790 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL6849 |
| Series (1) |
| GSE11404 |
Microarray analysis of pig innate immune responses after experimental infection with Actinobacillus pleuropneumoniae |
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