4 rats from each strain was exposed to an aerobic interval training program. Briefly, after 10 minutes of warm-up, rats ran uphill (25°) on a treadmill for 1.5 hours, alternating between 8 minutes at an exercise intensity corresponding to 85-90% of VO2max, and 2 minutes active recovery at 50 - 60%. Exercise was performed 5 days per week over 8 week; controls were age-matched rats that remained sedentary. We measured VO2max every week in exercising rats to adjust speed in order to maintain the intended intensity throughout the experimental period. The VO2max-testprotocol consisted of 20 minutes warm-up at 50-60% of VO2max, whereupon treadmill velocity was increased by 0.03 m/s every 2 minute until VO2 plateau despite of increased workload. The animals in the sedentary groups were treated similar to the exercise groups, except being exposed to exercise training and VO2max-tests. At approximately 7 months of age, and 48 hours after the last exercise session all the animals were sacrificed. One of the soleus muscles was formalin fixated for immunohistochemistry and morphological studies, whereas the other was snap frozen in liquid nitrogen and stored at -80oC for later genetic screening and protein analysis.
Extracted molecule
total RNA
Extraction protocol
Tissue samples (20 mg) were homogenized in 100 µL TRIzol (Life Technologies, Gaithersburg, MD) using a Mixer Mill MM301 (Company, City, State/Country) at 20-25 Hz. RNA clean-up was performed using RNA Mini kit (Qiagen, Germantown, MD). Total RNA was isolated and RNA clean up was performed according to the manufacturer's instructions. RNA integrity, purity and quantity were assessed by Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Nanodrop (NanoDrop Technologies, Baltimore, MD). The concentration of total RNA was measured by Nanodrop with ultraviolet spectrophotometry at 260/280 nm. RNA quality was assessed by electrophoresis on Bioanalyzer chips (Agilent Technologies). High quality RNA was classified as a 260/280 ratio above 1.8. Only samples with a 260/280 ratio between 1.8-2.2 and no signs of degradation were used for analysis.
Label
biotin
Label protocol
According to manufacture's instructions.
Hybridization protocol
According to manufacture's instructions. Labeled cRNA was prepared and hybridized to the RAE 230 2.0 chip from Affymetrix GeneChip (Affymetrix, Santa Clara, CA) comprised of 31,042 probe sets. On the Affymetrix GeneChip arrays, each gene is represented by a set of 11-20 probe pairs consisting of a perfect match (PM) and a mismatch (MM) probe. The statistical analysis is based on summary expression measures for each probe set.
Scan protocol
According to manufacture's instructions.
Description
We used rats artificial selected for high and low aerobic capacity, starting from the N: NIH stock obtained from the National Institutes of Health (USA). Briefly, the rats in each generation were tested for exercise capacity by treadmill running at 11 weeks of age. The individuals with the highest and lowest running capacity were selected and each group served as the mating population for the next generation. Female rats from generation 16 were used in this study. The study includes four groups; LCR trained (LCR-T) (n=4), LCR sedentary (LCR-S) (n=4), HCR trained (HCR-T) (n=4) and HCR sedentary (HCR-S) (n=4). Experimental protocols were approved by the respective Institutional Animal Research Ethics Councils
Data processing
Computing summary measures (RMA)
The summary measure for each probeset is computed based on a linear statistical model for background-corrected, normalized and log-transformed PM values for each probe pair by use of the robust multiarray average (RMA) method. The PM values are normalized using the quantile normalization method, normalizing the arrays such that the empirical distribution of the expression measures is equal across arrays.
Statistical analysis for finding differentially expressed genes
For each gene (probeset), a linear regression model, including parameters representing the effect of aerobe capacity is specified. Based on the estimated effects, tests for significant differential expression are performed using moderated T-tests.
To account for multiple testing, we calculated adjusted p-values controlling the False Discovery Rate (FDR), with the use of the Benjamini-Hochberg step-up procedure. Consequently, selecting differentially expressed genes based on a threshold of 0.05 on the adjusted FDR p-values means that the expected proportion of genes falsely classified as differential expressed should be below 0.05.
All statistical analyses on the gene expression data are performed using the R language (R Development Core Team, 2004) and packages affy, affyPLM and limma from the Bioconductor project.
