Animals were slaughtered in accordance with European regulations at Meadow Meats Limited, Rathdowney, Co Laois. Samples from the M. longissimus dorsi (LD) muscle were collected within 20 min postmortem. Muscle was stored in RNAlater™, (Ambion Ltd., Cambridge, UK) for 24 hr and transferred to -80°C for long term storage.
Growth protocol
Charolais × Limousin crossbred heifers were randomly assigned to one of two production systems a pasture-fed group (G) and a concentrate-fed group (C).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol® reagent (Sigma-Aldrich Corp., St. Louis, MO, USA) and Tissue Lyzer ™ (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The extracted RNA was treated with DNase I (Qiagen, Hilden, Germany) at room temperature for 10 min to remove genomic DNA. Total RNA was quantified and assessed for purity on a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples with a 260/280 ratio ≥ 2.0 were considered suitable for cDNA synthesis. Total RNA integrity number (RIN) was assessed on the Agilent 2100 Bioanalyser version A.02.12 (Agilent Technologies Inc., CA, USA) using an RNA 6000 Nano LabChip kit (Caliper Technologies Corp. MA, USA).
Label
biotin
Label protocol
Five μg of the amplified single-stranded cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies, San Carlos, CA) in accordance with manufacturers guidelines. The enzymatically and chemically fragmented products (50–100nt) were labeled via the attachment of biotinylated nucleotides onto the 3' end of the fragmented cDNA.
Hybridization protocol
The fragmented and labeled cDNA was added to the hybridisation cocktail in accordance with the NuGEN guidelines. Following hybridisation for 18 hr at 45°C, the array was washed and stained on the GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA), inserted into the Affymetrix autoloader carousel and scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Scan protocol
Following hybridisation for 18 hr at 45°C, the array was washed and stained on the GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA), inserted into the Affymetrix autoloader carousel and scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Data processing
The microarrays were processed using a custom pipeline written in R using bioconductor libraries. The arrays were read in using functions from simpleaffy, pre-processed and normalised using the Factor Analysis for Robust Microarray Summarization (FARMS) algorithm (Hochreiter et al., 2006). The data was normalised using the quantile normalisation technique as implemented in the qFarms function. The normalised probeset data was then filtered to retain only the informative probes as determined by FARMS.
