treatment: cells were pretreated with 0.1% DMSO (vehicle, control) for 96h prior to harvesting. background: caucasian gender: female age: 50 years tissue: muscle
Treatment protocol
Experiments were performed after 7 days of differentiation. Cells were pretreated for 96 h with 0.1% DMSO (vehicle, control) or the PPARδ agonist GW501516 (10 nmol/l) prior to harvesting.
Growth protocol
Human skeletal muscle cells (myotubes) grown from satellite cells were isolated from the M. obliquus internus abdominis of 4 healthy female donors. Body mass index, fasting glucose, insulin, plasma lipids and blood pressure of donors were within normal range. The biopsies were obtained with informed consent and the procedure was approved by the National Committee for Research Ethics (Oslo, Norway). The cells were cultured in DMEM-Glutamax (5.5 mM glucose), 2 % FCS, 2 % Ultroser G, P/S, and amphotericin B. At 70-80 % confluence the growth medium was replaced by DMEM-Glutamax™ supplemented with 2 % FCS, P/S, 1.25 µg/ml amphotericin B, and 25 pM insulin to induce differentiation. The cells were cultured in humidified 5 % CO2 atmosphere at 37 °C, and the medium was changed every 2–3 days.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared using the Agilent Total RNA isolation kit. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
Hybridization protocol
Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 fluidics station using the protocol FS450_0001, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.20.4).
PPARδ activation in human myotubes increases mitochondrial fatty acid oxidative capacity and reduces glucose utilization by a switch in substrate preference.
