One or two genes were selected from interesting functional categories revealed by the microarray analysis as significantly differentially regulated by RE. Following completion of the microarray experiment, muscle biopsy samples were only selleck Dasatinib available for a subset of subjects. Total RNA was isolated from these sam ples with TRIzol reagent. The quality and quantity of the total RNA samples was checked using a spectrophotometer prior to reverse transcription. The OD 260 280 ratio of our samples ranged from 1. 84 1. 97 indicating good quality. Random hexamers were used to generate single strand cDNA using MMLV Reverse Transcriptase according to the manufacturers protocol. cDNA was cleaned using QIAquick PCR purification kit.

The qRT PCR was performed using an Applied Biosystems Taqman Gene Expression Assay containing a FAM dye labeled Taqman MGB probe on the Applied Biosystems 7500 Real Time PCR System. The selected genes and TaqMan assays are displayed in supplementary data. The Inhibitors,Modulators,Libraries reaction was prepared according to the standard Taqman gene expression assay protocol in a total volume of 20 ul. Thermo cycling conditions were standard and as follows, 10 min at 95 C followed by 40 cycles of 15 s at 95 C and 1 min at 60 C for annealing. All samples were analyzed in duplicate. Eukaryotic beta 2 microglobulin was included as an internal control to calibrate the expres sion levels of target gene. The validity of B2 M as an internal control in acute resistance exercise studies in human skeletal muscle has been previously tested and confirmed.

After amplification, all data were normal ized to B2M, and the relative changes in gene expres sion between exercised and control muscle was calculated Inhibitors,Modulators,Libraries using the 2^Ct method. As a prere quisite assumption of using the 2^Ct method for analyses, the consistency GSK-3 of the amplification efficiency of B2 M and all the target genes were tested via serial dilution and confirmed in two randomly selected samples. To validate the microarray data, correlation was tested for fold changes measured by microarray and qPCR for all the selected genes. For the genes represented with multiple probes in microarray, the probe with the high est average signal intensity was used. Since the data was not normally distributed, Spearmans Rho was used. For the microarray, the data input into the correlation analysis was the weighted average of the fold change for each gene on the array representing all replicate individuals.

For qRT PCR, we used the mean fold change calculated as 2^Ct for all replicate individuals. Inhibitors,Modulators,Libraries Statistical analysis Data on physical characteristics of subjects are reported as mean SE or median, sex differ ences were tested either Inhibitors,Modulators,Libraries by t test, or by Wilcoxon Rank Sum Test when the assumption of normality was vio lated, and statistical significance Gefitinib chemical structure was set at p 0. 05.