The low-RFI (RFIneg) and high-RFI (RFIpl) pigs were fed ad libitum with high fat ,high fiber (HF) diet or low fat, low fiber (LF) diet.
Extracted molecule
total RNA
Extraction protocol
For total RNA extraction, samples of frozen tissues (80 to 100 mg each) were homogenized in Trizol reagent (Invitrogen, Cergy-Pontoise, France) using a Precellys homogenizer (Ozyme,Saint-Quentin-en-Yvelines) and were then treated as previously described (Vincent et al., 2012). RNA was finally purified using a silica-membrane technology under vacuum (Nucleospin 8 RNA kit, Macherey Nagel, Hoerdt, France)
Label
Cy3
Label protocol
Total RNA (150 ng) from each sample was labeled with Cy3 dye using the low RNA input linear amplification kit (Agilent Technologies, Les Ulis, France) following the manufacturer’s instructions. Briefly, a two-step procedure was used to generate fluorescent complementary RNA (cRNA) by using T7 RNA polymerase. Samples were then purified with an RNeasy mini elute kit (Qiagen, Hilden, Germany)
Hybridization protocol
Microarray hybridizations were carried out in Agilent’s SureHyb hybridization chambers containing 600 ng of Cy3-labeled cRNA sample per hybridization. Hybridization reactions were performed at 65°C for 17 h using Agilent’s Gene Expression Hybridization Kit
Scan protocol
After washing, microarrays were scanned at 3 µm/pixel resolution using the Agilent DNA Microarray Scanner G2505C, and images were analyzed with Agilent Feature Extraction Software (Version 10.7.3.1, protocol GE1_107_Sep09)
Description
Gene expression in pig skeletal muscle with high residual feed intake fed with low fat diet
Data processing
All analyses were then performed using the R software version 2.10.0 (R Development Core Team, 2008). Raw spots intensities were first submitted to quality filtration based on 4 criteria: intensity, uniformity, saturation and outliers detection. Intensities of filtered spots were transformed into log2(Cy3), and data were normalized by median centering, i.e. subtracting the median value across all probes from all raw values for each sample to obtain experimentally consolidated gene expression values. Normalized data were submitted to an analysis of variance using the fixed effects of genetic line breed (RFIpl or RFIneg), diet effect (HF or LF) and the interaction between line and diet.
