RNeasy Maxi Kit with DNase treatment following the enclosed protocol (Qiagen)
Label
Alexa-647
Label protocol
20 µg total RNA was labelled with Alexa-647 using the Superscript Plus Direct cDNA Labeling System (Invitrogen) following the enclosed protocol. Spike-in RNA (red) from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions.
Breed: Hampshire, gender: gilt, status: healthy, age: 4 to 6 months, tissue: pool of all RNA extractions
Extracted molecule
total RNA
Extraction protocol
RNeasy Maxi Kit with DNase treatment following the enclosed protocol (Qiagen)
Label
Alexa-555
Label protocol
20 µg total RNA was labelled with Alexa-555 using the Superscript Plus Direct cDNA Labeling System (Invitrogen) following the enclosed protocol. Spike-in RNA (green) from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions.
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 prepares 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: GenePix Autoloader 4200AL, 5 µm resolution, 75 % laser power and PMT adjusted individually for each channel. Image analysis software: GenePix Pro (version 6.0.1.22, Molecular Devices) using FeatureType = Irregular Filled and BackgroundSubtraction = MorphologicalClosingFollowedByOpening
Description
Statistical analysis was carried out in the R computing environment version 2.6 using the package Linear Models for Microarray Analysis (Limma, version 2.9.17) which is part of the Bioconductor project. The log2-transformed ratios of Alexa-647 to Alexa-555 were normalized within-slide using the global-loess method with default parameters as implemented in Limma.
Data processing
Muscle tissue samples from heart (HEA), Vastus intermedius (VIN), Infraspinatus (ISP), Supraspinatus (SSP), Biceps femoris (BFE), Longissimus dorsi (LDO), Semimembranosus (SME), Semitendinosus (STE) and Triceps brachii (TBR). A reference sample was constructed by combining all samples. The exact same tissue samples were used for expression profiling with cDNA microarrays and 70-mer long oligonucleotide microarrays.
