Breed: Danish Landrace/Yorkshire/Duroc cross, Herd: high health free from A. pleuropneumoniae, Gender: castrated males, Age: 8-10 weeks, Treatment: AP serotype 5b infected, Sample: Lung_infected_visually-unaffected-area, Animal: 34
Biomaterial provider
Technical University of Denmark, National Veterinary Institute, Denmark
Treatment protocol
Pigs assigned as healthy non-inoculated control animals were killed and sampled prior to inoculation of the infected group. Samples of approximately 500 mg tissue were taken from the lungs, snap frozen in liquid nitrogen and stored at minus 80 degrees until further use. Pigs assigned as infected animals, were inoculated with Actinobacillus pleuropneumoniae 5b (strain L20) by dripping 1 ml bacterial suspension, (McFarland 0.5, mixed 1:1 with Brain Heart Infusion + 0.1 % NAD) containing approximately 0.7 x107 CFU, in each nostril during inhalation. Culture of bacteria as well as inoculation procedures has previously been described by (Kristensen et al., Vet. Microbiol. 98, 243-249, 2004). Animals were killed 14 – 18 hours post inoculation by means of captive bolt pistol followed by pitching and exsanguinations. All animal procedures were approved by the Danish Animal Experiments Inspectorate. Immediately after killing, lungs were inspected for sizes and numbers of pulmonary lesions. To verify the infection, samples from lung, liver, tonsil and spleen were cultivated on PPLO agar (BD, Brøndby, Denmark) to re-isolate the inoculation strain, which hereafter was serotyped using latex agglutination (Giese et al., Acta. Vet. Scand. 34, 223-225, 1993). From the lungs of infected animals, samples of approximately 500 mg tissue were taken from three different sites, 1. necrotic area, within the pulmonary lesion, 2. demarcation zone, surrounding area to the pulmonary lesion, and 3. visually unaffected area, as far away from the pulmonary lesions as possible. Samples were snap frozen in liquid nitrogen and stored at minus 80 degrees until further use.
Extracted molecule
total RNA
Extraction protocol
RNeasy Midi Kit with DNase treatment following the enclosed protocol (Qiagen)
Label
Alexa-555
Label protocol
20 µg total RNA was labelled by using the Superscript Direct cDNA Labeling System (Invitrogen) following the enclosed protocols. Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the labelling reactions. Green spike-in RNA was added to the common reference samples and red spike-in RNA was added to the samples. The fluorescently labelled cDNA was purified using the NucleoSpin 96 Extract II PCR Clean-up kit (Macherey-Nagel, Düren, Germany).
Channel 2
Source name
Reference, pool of equal amount of total-RNA from all samples
Breed: Danish Landrace/Yorkshire/Duroc cross, Herd: high health free from A. pleuropneumoniae, Gender: castrated males, Age: 8-10 weeks, Reference, pool of equal amount of total-RNA from all lung samples in experiment
Biomaterial provider
Technical University of Denmark, National Veterinary Institute, Denmark
Treatment protocol
Pigs assigned as healthy non-inoculated control animals were killed and sampled prior to inoculation of the infected group. Samples of approximately 500 mg tissue were taken from the lungs, snap frozen in liquid nitrogen and stored at minus 80 degrees until further use. Pigs assigned as infected animals, were inoculated with Actinobacillus pleuropneumoniae 5b (strain L20) by dripping 1 ml bacterial suspension, (McFarland 0.5, mixed 1:1 with Brain Heart Infusion + 0.1 % NAD) containing approximately 0.7 x107 CFU, in each nostril during inhalation. Culture of bacteria as well as inoculation procedures has previously been described by (Kristensen et al., Vet. Microbiol. 98, 243-249, 2004). Animals were killed 14 – 18 hours post inoculation by means of captive bolt pistol followed by pitching and exsanguinations. All animal procedures were approved by the Danish Animal Experiments Inspectorate. Immediately after killing, lungs were inspected for sizes and numbers of pulmonary lesions. To verify the infection, samples from lung, liver, tonsil and spleen were cultivated on PPLO agar (BD, Brøndby, Denmark) to re-isolate the inoculation strain, which hereafter was serotyped using latex agglutination (Giese et al., Acta. Vet. Scand. 34, 223-225, 1993). From the lungs of infected animals, samples of approximately 500 mg tissue were taken from three different sites, 1. necrotic area, within the pulmonary lesion, 2. demarcation zone, surrounding area to the pulmonary lesion, and 3. visually unaffected area, as far away from the pulmonary lesions as possible. Samples were snap frozen in liquid nitrogen and stored at minus 80 degrees until further use.
Extracted molecule
total RNA
Extraction protocol
RNeasy Midi Kit with DNase treatment following the enclosed protocol (Qiagen)
Label
Alexa-647
Label protocol
20 µg total RNA was labelled by using the Superscript Direct cDNA Labeling System (Invitrogen) following the enclosed protocols. Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the labelling reactions. Green spike-in RNA was added to the common reference samples and red spike-in RNA was added to the samples. The fluorescently labelled cDNA was purified using the NucleoSpin 96 Extract II PCR Clean-up kit (Macherey-Nagel, Düren, Germany). The labelled reference samples were mixed and divided into aliquots before combining it with a labelled sample.
Hybridization protocol
The slides were hybridized in a Discovery XT hybridization station (Ventana Discovery Systems, Tucson, AZ, USA). Transfer Chip Prep-2 from 4 ºC to room temperature 1 hour before use. Prepare ChipSpread by mixing equal volumes of ChipSpread A (20 mg/mL BSA, 4x SSC, 0.5 mg/mL sodium azide) and B (formamide; 2 mg/mL SDS) and incubate at room temperature for 1 hour before use. A total of 2.5 mL is needed per slide. Print labels, trim them and place them on the slides. Mix the Chip Map reagents (Chip Prep-1, -2 and - 3) by inversion, remove the cap and place the reagents in the Discovery. Place the slides in the machine and initiate the run. Cover slide with 2.5 mL ChipSpread when the message appears (after few minutes). The machine now runs for app. 1.5 hours to pre-hybridize the slides. Heat a waterbath to 90°C or use a PCR machine. Mix the Chiphybe80, add 200 µL to the sample (<20 µL) and mix carefully. Heat the sample mixture at 90°C for 3 minutes and mix carefully by pipetting. Press the button on the machine which then prepares the slides for hybridization. When the message appears apply the samples onto the slides and press the button and the machine hybridizes at 48 ºC for 6 hours. Wipe oil from backside of slides using a clean-room napkin and place slides in the slide-holder from the High Throughput Wash Station (Telechem, cat.no. HTW) placed in a mTub filled with RiboWash. If processing more than 20 slides, place equal number of slides in two slide-holders and continue in parallel. Transfer the slide-holder to a HTW filled with RiboWash and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with RiboWash and repeat the wash. Dip the slide-holder in 2x SSC filled in a mTub (200 mL 20x SSC, Elga H2O + 1800 mL water). Transfer the slide-holder to a HTW filled with 2x SSC and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with 2x SSC and repeat the wash. Dip the slide-holder 10 times in 0.1x SSC filled in a mTub (5 mL 20x SSC, Elga H2O + 995 mL water) and leave the holder submerged in 0.1x SSC. Transfer the slides to a mBox slide holder placed in a mTub filled with Elga H2O. Dry arrays by centrifugation (at 300 x g for 4 min placed in a mBox).
Scan protocol
Scanner: ScanArray Express HT system (Perkin Elmer), 5 µm resolution, 100 % laser power and PMT adjusted individually for each channel. Image analysis software: GenePix Pro (version 6.0.1.27, Molecular Devices) using FeatureType = Irregular Filled and MorphologicalClosingFollowedByOpening as background measurement.
Description
Expression profiles of porcine lung tissues were studied to inditify genes beeing significantly affected at various sites in bacterially infected porcine lungs as opposed to not infected porcine lungs. Samples was taken from three different sites of the lung in pigs experimentally infected with A. pleuropneumoniae, serotype 5b, these were; 1. Necrotic area, samples were taken from within the pulmonary lesion, 2. Demarcation zone, samples were taken from the suburb area of the pulmonary lesion, and, 3. Visually unaffected area, samples were taken either from the visually unaffected lung lobules, if there is one, or as far away from the pulmonary lesion in an affected lung. Samples of lung tissue were furthermore sampled from healthy non-inoculated control pigs from the same heard. Samples were investigated for changes in gene expression by means of quantitative real-time PCR and global cDNA microarrays.
Data processing
Statistical analysis was carried out in the R computing environment (version 2.6.1 for Windows) using the package Linear Models for Microarray Analysis (Limma, version 2.12.0) which is part of the Bioconductor project. Control spots and spots with more than 50 % of saturated pixels were weighted 0 before the log2-transformed ratios of Alexa-555 to Alexa-647 (not background corrected) were normalized within-slide using global-loess with default parameters as implemented in Limma. The set of normalized log-ratios were analyzed in Limma to identify genes being significantly differentially expressed between lungs of uninfected control animals and the three different regions sampled from lungs of infected animals (unaffected areas, the demarcation zone, and necrotic areas). The false discovery rate was controlled using the method of Benjamini and Hochberg as implemented in Limma and a corrected P-value below 0.01 was considered significant.
Transcriptional profiling at different sites in lungs of pigs during acute bacterial respiratory infection
Data table header descriptions
ID_REF
Unique spot identifier
VALUE
background corrected and lowess-normalized log2 ratios (555/647)
Alexa 555 Median
Alexa-555 (channel 2) median feature intensity
Alexa 647 Median
Alexa-647 (channel 1) median feature intensity
Weight
binary quality spot weight applied before normalisation, assigning a weight of 0 to Control spots, spots with a SNR value below 1, and spots with more than 50 % of saturated pixels
