|
| Status |
Public on May 04, 2007 |
| Title |
DFVF1_pig3_lung_infected |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lung tissue, infected
|
| Organism |
Sus scrofa |
| Characteristics |
Animal: 10-12 week old castrate of Danish Landrace/Yorkshire/Duroc from a high health herd (Specific Pathogen Free)
Tissue: lung
State: infected with Actinobacillus pleuropneumoniae
|
| 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)
|
| |
|
| Channel 2 |
| Source name |
Common reference
|
| Organism |
Sus scrofa |
| Characteristics |
Mixture of total RNA purified from infected and healthy pig lung 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)
|
| |
|
| |
| Hybridization protocol |
The slides were pre-hybridized (5 x SSC, 0.1 % SDS, 1 mg/mL BSA, 0.22 µm filtrated) at 42 ºC for 45 min and washed (2x5 dips in water) followed by a dip in isopropanol and centrifugation (300 x g). mSeries LifterSlip (25x60 mm, Erie Scientific Company) were cleaned by dipping in water followed by several dips in ethanol and left for drying. A lifterslip was placed on each slide and incubated at 42 ºC to preheat. The hybridization chamber, a 6x17x22 cm lidded plastic box with a stainless steel “table” and a napkin wetted with hybridization buffer, was placed at 42 ºC to preheat. The purified labeled cDNA were combined pair wise and 20 µg Poly-dA (Invitrogen), 25 µg yeast tRNA (Invitrogen) and 20 µg pig CoT-DNA was added. The samples were speedvaced until dry (medium heat, app. 30 min), resuspended in 70 µL hybridization buffer (50 % formamide, 5x SSC, 0.1 % SDS), incubated at 95 ºC for 3 min, snap cooled on ice and centrifuged. The samples were applied to the pre-heated slides and placed in the hybridization chamber which was sealed and incubated at 42ºC for 12 to 16 hours. Following hybridization, each array was immersed in 100 mL wash-1 (2x SSC, 0.1% SDS, 0.22 µm filtrated) at 42ºC until the lifterslip moves freely away from the slide. The arrays were transferred to a mBox (Erie Scientific Company) and immersed in wash-1 (2x SSC, 0.1% SDS, 0.22 µm filtrated) at 42ºC for 5 min., in wash-2 (0.1x SSC, 0.1% SDS, 0.22 µm filtrated) at ambient temperature for 10 min., 4 times in wash-3 (0.1x SSC, 0.22 µm filtrated) at ambient temperature for 1 min., rinsed in wash-4 (0.01x SSC, 0.22 µm filtrated) for 5 to 10 sec., dipped 5 times in water and dried by centrifugation (300 x g).
|
| Scan protocol |
Scanner: ScanArray Express HT system (Perkin Elmer), 10 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 lung tissue was extracted from healthy pig lung tissue and from lung tissue infected 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_27K2_v2 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.
|
| |
|
| Submission date |
Mar 29, 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 |
GPL3594 |
| Series (1) |
| GSE4577 |
Early pig response to experimental infection with Actinobacillus pleuropneomuniae |
|
