erase-2 Aldehyde dehydrogenase 1 family member A1 Trifunctional protein b subunit Myosin Heavy Chain I Myosin Heavy Chain IIa Myosin Heavy Chain IIx PGC-1alpha PPARdelta Myostatin Forward Primer ggctatccagcgtactccaa actgaggtccaccctgactac gaaaggcacctgcggtatt tcatcacctttcctctggatac ggcaggagaagcaagatga cgccagacttacctgtcctact aaacaagcaatgtggctagaga cctggaacatctggagacct caatctagctaaattccgcaagc aaatggtggaaagaagagagtcc ttgctaaacgactccgagaa actgagttcgccaagagcat gacccgtcgagactcctaca Reverse Primer gatgaaacccagacacatagca tcgcattcttaggcttctca catgccgttctttgttctgta agaatggtgcccatcacac gcaccattgaaggaacctatg ctcctcagttgcaggattaaag ggcttggttggcagagatac agctgctttcggaccttct tcacttatgacttttgtgtgtgaacct aatacagcttcatccagggc tgcaaagttccctctctgct gtgcacgccatacttgagaa aataccagtgcctgggttca All primer sequences are shown 59 to 39, left to right. doi:10.1371/journal.pone.0006335.t002 1% CaCl2 and 2% CoCl2 for 3 min, and then incubated in 1% ammonium sulphide for 30 s at room temperature. Sections were photographed at 2006magnifications with a microscope in conjunction with a SPOT digital camera. Based on the staining intensity at pH 4.60, the three fiber types were classified as Type I, IIa and IIx. Cross-sectional areas of the muscle fibers were determined using an image analysis program. Alta, CA) and analyzed using GeneChip Analysis software. Statistical Analysis of MicroArray The statistical analysis was based on the Affymetrix signal. Exploratory statistical tools were used to check data quality. There were no quality control problems with the data. The data was then filtered based on the given algorithms for gene content levels, 1975694 which filtered out genes that have low content levels compared to background. When a BMS-345541 chemical information minimum of 19 out of 24 gene replicates read as Affymetrix Absent Calls, the gene is filtered out. An ANOVA model with log of the Affymetrix signal as a response is fitted for each one of the genes that are not filtered based on Affymetrix Absent Calls. Significant differential content is calculated by the NLOGP ). A gene was considered to be differentially expressed if the NLOGP measure was greater than the 4, and the fold change was at least 1.2. Microarray Preparation Muscle samples from study 1 were used to extract RNA, and the RNA was prepared according to Affymetrix recommendations. In brief, muscle samples were homogenized in TRIzol Reagent by shaking in a mixer mill with tungsten carbide beads as recommended by the manufacturer. RNA was purified using an RNeasy Mini Kit. Ten micrograms of purified RNA was reverse transcribed using SuperScript II Reverse Transcriptase and T7-24 primer followed by second strand DNA synthesis as per the manufacturer’s instructions. Contaminants were removed from the samples by phenol-chloroformisoamyl 
alcohol extraction, 23321512 and the clean cDNA was recovered by ethanol precipitation, using a RNA Transcript Labeling Kit, it was further converted into biotin-labeled cRNA, as per manufacturer’s instructions. Moreover, cRNA was purified using a RNeasy Mini Kit and fragmented in pieces,200 bases by incubation in fragmentation buffer. Samples were stored at 220uC until hybridization. Samples were hybridized on human HG-U133 Plus 2.0 Arrays using protocols as recommended by Affymetrix. In brief, 24 gene chips were used to compare 12 women to 12 men. Biotinylated cRNA was hybridized for 16 h at 45uC in a GeneChip Hybridization Oven 640. Arrays were placed in a GeneChip Fluidic Station 400 for a series of washes, followed
