|
| Status |
Public on May 04, 2007 |
| Title |
DFVF1_pig1_liver_control |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver tissue, healthy
|
| Organism |
Sus scrofa |
| Characteristics |
Animal: 10-12 week old castrate of Danish Landrace/Yorkshire/Duroc from a high health herd (Specific Pathogen Free)
Tissue: liver
State: healthy
|
| Biomaterial provider |
Danish Institute for Food and Veterinary Research
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Maxi Kit with DNase treatment (Qiagen)
|
| Label |
Alexa 594
|
| Label protocol |
Superscript Indirect cDNA Labeling System (Invitrogen) and ARES cDNA labeling kit (Molecular Probes/Invitrogen). Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions. “Green” spike-in RNA was added to the common reference samples and “red” spike-in RNA was added to the samples.
|
| |
|
| Channel 2 |
| Source name |
Common reference
|
| Organism |
Sus scrofa |
| Characteristics |
Mixture of total RNA purified from challenged and healthy pig liver tissue
|
| Biomaterial provider |
Danish Institute for Food and Veterinary Research
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Maxi Kit with DNase treatment (Qiagen)
|
| Label |
Alexa 488
|
| Label protocol |
Superscript Indirect cDNA Labeling System (Invitrogen) and ARES cDNA labeling kit (Molecular Probes/Invitrogen). Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions. “Green” spike-in RNA was added to the common reference samples and “red” spike-in RNA was added to the samples.
|
| |
|
| |
| 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 “button” on the machine which then prepare the slides for hybridization. When the message appears apply the samples onto the slides and press “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 % LP and PMT adjusted individually for each channel.
Image analysis software: ScanArray Express, ver. 3.0 (Perkin Elmer) using the histogram method with default settings.
|
| Description |
RNA from liver tissue was extracted from healthy pigs and from pigs challenged with Actinobacillus pleuropneumoniae, reverse transcribed and labeled with Alexa 594. A common reference (equal amounts of RNA from all samples) was reverse transcribed and labeled with Alexa 488. Samples and reference were hybridized simultaneously to DIAS_PIG_55K2_v1 slides and fluorescence in the Alexa 594 and Alexa 488 channels was acquired using a microarray scanner.
|
| Data processing |
Statistical analysis was carried out in the R computing environment (ver 2.3.0, developmental version, for Windows) using the package Linear Models for Microarray Analysis (Limma, ver 2.4.11) which is part of the Bioconductor project. The log2-transformed ratios of Alexa-594 to Alexa-488 (not background corrected) were normalized within-slide using printtip-loess with default parameters as implemented in Limma and can be found in the “Value” column.
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| |
|
| Submission date |
Mar 30, 2006 |
| Last update date |
May 03, 2007 |
| Contact name |
Jakob Hedegaard |
| E-mail(s) |
Jakob.Hedegaard@ki.au.dk
|
| Phone |
(+45)89991363
|
| Organization name |
Aarhus University, Faculty of Agricultural Sciences
|
| Department |
Department of Genetics and Biotechnology
|
| Lab |
Molecular Genetics and System Biology
|
| Street address |
PO-box 50
|
| City |
Tjele |
| ZIP/Postal code |
DK-8830 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL3585 |
| Series (1) |
| GSE4577 |
Early pig response to experimental infection with Actinobacillus pleuropneomuniae |
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