Microarray significance.1.2 fold increase or decrease. NLOGP, negative log of the p value,.4.0. N = 12 men and 12 women. doi:10.1371/journal.pone.0006335.t003 RNA isolation for RT-PCR Muscle biopsy samples from study 2 were used to isolated RNA as described previously. Briefly, frozen muscle was thawed in a Tenbroeck homogenizer with 1 ml of TRIzol reagent and homogenized on ice. The STA 9090 custom synthesis homogenate was extracted with 200 ml of chloroform. The aqueous phase was removed and the RNA was precipitated at room temperature using 500 ml of iso-propanol and washed twice with 75% ethanol. The final RNA pellet was air dried at room temperature and resuspended in 14 ml ddH2O and treated with DNaseI. The RNA samples were quantified by spectrophotometer and the quality was assessed by agarose gel electrophoresis. TaqManH Real-time RT-PCR TaqManH real-time RT-PCR was conducted on total RNA. Duplex RT-PCR was performed using an iCycler real-time PCR Sex Difference in 
mRNA Content system. One-step TaqManH RT-PCR Master Mix Reagent was combined with RNA, target gene primers and probes, and internal standard gene primers and probes in the same reaction. Specific primers and probes to each target gene were designed based on the cDNA sequence in GenBank with primer 3 designer. Their specificity was monitored using Blast. Primer sequences are listed in to horseradish peroxidase and specific antibody binding was detected using the chemiluminescence detection reagent ECL+. Scanned films 20347963 were analyzed using ImageJ 1.40 software. Statistical analysis All statistical analyses, for mRNA content of the genes, was performed on linear data 22CT for evaluation of internal standards, 22DCT for target gene normalized with internal reference. Data on sex differences about target gene mRNA content were analyzed using a Student’s t test. All results from evaluation of target gene are expressed as mean6SEM, using 22DCT. Western blot data was normalized by the loading control and a Student’s t test was preformed to test for a difference between men and women. Data are presented as means6SEM. The data regarding muscle fiber composition were analyzed using a single factor ANOVA and expressed as mean6SEM. All analyses were done using statistics software. Statistical significance was set at a#0.05. Western blot analysis Thirty mg of tissue was used for protein content analysis. Muscle tissue was homogenized in a phosphate lysis buffer; 50 mM K2HPO4, 1 mM EDTA, pH 7.4, 0.1 mM DDT, PhosSTOP, Protease inhibitor cocktail tablets. Protein concentrations were calculated by Bradford assay and equal amounts of protein were boiled in Laemmli buffer, 18316589 resolved by SDS-PAGE, transferred to nitrocellulose paper and immunoblotted with desired antibodies. Primary antibodies; HADHB, ACAA2, catalase, PPARd, MHC I, MHC II and b-actin were all purchased from. Secondary antibodies conjugated Results Sex alters mRNA content in skeletal muscle; microarray The mRNA abundance in skeletal muscle between men and women was significantly different for 66 genes, after Y-linked genes were removed. Of these 66 genes, 49 genes have known functions in metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Conversely, 17 of the genes identified in the current analysis do Sex Difference in mRNA Content not have known functions. Of the 49 genes with known function, women had higher content of 25 genes, compared to men. Subse
