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.
