Human gene 1.0 st

The list of differentially genes in the present sprint study was compared to a list of genes from a ‘control’ experiment in order to find overlapping genes. Biopsies were performed with an interval of 2.5 h in a non-exercised and fasting condition [15 (link)]. We obtained normalized and processed data files from Vissing et al [15 (link)] and cut-off values for fold change and FDR were chosen to be 1.2 and 10%, i.e. the same as in the present study. Thirty-nine upregulated and 52 downregulated genes were identified. The selected study was chosen to match the study populations as far as possible with regard to subject characteristics (6 healthy young men), the time interval between the biopsies (2.5 h) and array platform (Affymetrix Human Gene 1.0 ST). Statistical significance of overlap was analysed by using the hypergeometric test. Seven genes were upregulated and 14 genes were downregulated in common between controls and sprint exercise (S5 Table).

Whole genome gene expression of the samples was measured by the Human Gene 1.0 ST Affymetrix microarray. 300 ng of total RNA were used for microarray analysis following the manufacturer’s instructions. Both the WT Expression Kit by Ambion and the GeneChip Hybridization Wash and Stain kit by Affymetrix were used in the process. Briefly, during the three-day protocol, complementary single-strand DNA was synthesized from RNA, to be later fragmented, labeled and hybridized during 16 hours. The hybridized microarrays were washed and stained in a GeneChip Fluidics Station 450 and scanned in a GeneChip 7G Scanner afterwards.
The data of the .cel files were normalized with the Robust Multichip Average (RMA) in the Expression Console software by Affymetrix. As multiple-testing correction makes the group-wise comparisons more stringent as more genes are included in the analysis, we filtered the data to remove the least informative genes using the BRB-Array Tools software [BRB-ArrayTools Development Team, version 4.2.1] implemented in Microsoft Excel [Microsoft Corporation, Microsoft Office Professional Edition 2003]. The genes with a 90^th percentile value smaller than 55.7 (the mean intensity of the negative control probes) were removed and 6451 probesets were left for the subsequent analysis.

Whole genome gene expression of the samples was measured by the Human Gene 1.0 ST Affymetrix microarray. First, RNA integrity was checked with the Agilent RNA 6000 Nano kit and the samples with an RNA Integrity Value (RIN) above 6 were accepted to be further processed. 300 ng of total RNA were used for microarray analysis following the manufacturer’s instructions. Both the WT Expression Kit by Ambion and the GeneChip Hybridization Wash and Stain kit by Affymetrix were used in the process. Briefly, during the three-day protocol, complementary single-strand DNA was synthesized from RNA, to be later fragmented, labeled and hybridized during 16 hours. The hybridized microarrays were washed and stained in a GeneChip Fluidics Station 450 and scanned in a GeneChip 7G Scanner afterwards. Two of the relapse samples failed to provide any signal and, thus, the 73 files created from the scanning were stored for subsequent analysis.