Ten young healthy Caucasian men (24-27 years) were recruited to the study. Subjects were requested to abstain from strenuous physical activity and alcohol consumption three days prior to the examination. To ensure standardized conditions, all subjects were provided with a nutritionally standardized diet three days prior to the first experimental day and during the bed rest with adjusted caloric content to ensure weight stability. All subjects were admitted to Steno Diabetes Center for ten days and were not permitted to deviate from a half-recumbent position during this period. Toilet visits, limited to a total of 15 minutes per day, were allowed. Study subjects were allowed to use a laptop computer, watch television and read in the bed. Body weight of all subjects was recorded every morning throughout the intervention to ensure weight stability. After bed rest a DXA scan and a VO2max test was again performed. For ethical reasons and for the purpose of studying reversibility of changes in DNA methylation, gene expression and protein levels, all subjects completed a four-week retraining program after the 9 days of bed rest. The program consisted of supervised cycle ergometry 30 min/day, 5 days/week on 70% of subjects’ VO2max
Extracted molecule
total RNA
Extraction protocol
Skeletal muscle samples were collected in all participants in both the basal and insulin stimulated state before and after bed rest, but only in the basal state after four weeks of retraining. Muscle biopsies were collected from the vastus lateralis muscle using a Bergstrom needle with suction under local anesthesia (1% lidocaine). Biopsies were immediately frozen in liquid nitrogen and transferred to a -80ºC freezer. Extraction of total RNA and genomic DNA from the muscle biopsies was performed with TRI reagent (Sigma-Aldrich, St. Louis, MO, USA). Both RNA and DNA were quantified by spectrophotometric analysis and RNA used for the microarray analysis was quality-assessed by the Agilent Bioanalyser 2100 using the RNA 6000 Nano Assay Kit (Agilent Technologies, Wokingham, Berkshire, UK).
Label
Cy3
Label protocol
RNA from muscle tissue and Agilent One-Color RNA Spike-In RNA were labeled with reagents supplied in the Agilent Quick Amp Labeling Kit, One-Color. The labeled cRNA was then purified with the RNeasy Mini Kit (Qiagen Ltd, Crawley, UK).
Hybridization protocol
The labeled cRNA was hybridized using Agilent's Hybridization Kit to the Whole Human Genome Oligo Arrays. The slides were then washed in Wash Solution 1 (0.005% Triton X-102), Wash Solution 2 (0.005% Triton X-102) acetonitrile and finally contained in Agilent's Stabilization and Drying Solution
Scan protocol
The slides were scanned with the Agilent G2565BA Microarray Scanner System. For data extraction and quality control, the Agilent G2567AA Feature Extraction Software (v.10.5.1.1) was used.
Description
Gene Expression analysis of vastus lateralis muscle, post inactivity, basal
Data processing
Whole genome microarray analysis was performed on RNA extracted from biopsies collected from the vastus lateralis muscle before bed rest in the basal (T0-B) and insulin-stimulated state (T0-I), after bed rest in the basal (T10-B) and insulin-stimulated state (t10-I) and in the basal state after 30 d retraining (T40-B). A comparison of basal gene expression between T0-B, T10-B and T40-B was performed to identify genes up or downregulated after bed rest, and to assess the effect of re-training on the expression of these genes. A second analysis compared gene expression changes in response to insulin before (T0-B v T0-I) and after (T10-B v T10-I) bed rest. The data were analyzed using Genespring GX 7.3 Expression Analysis software (Agilent Technologies). Text tab delimited raw files were imported into Genespring. Standard data transformation, chip and gene normalisations were applied, in addition to standard QC procedures. gProcessedSignal after normalization by shift to 75th percentile and baseline transformation to median of all samples is provided in the Sample data table below. Volcano plot analysis was used to identify differentially expressed genes in both comparisons, using both p<0.05 and </> 1.2 fold change filters. Differentially expressed gene lists were exported into Ingenuity Pathway Analysis (Ingenuity Systems) to identify common functional and signalling pathways represented in the gene lists.
