title: Illumina Genome Analyzer II sequencing; Transcriptomic analysis of anti-adipogenic effect of oleuropein in human mesenchymal stem cells
design: Transcriptomic analysis of anti-adipogenic effect of oleuropein in human mesenchymal stem cells
library_construction_protocol: Bone-marrow human mesenchymal stromal-cells were obtained from three donors recruited in the Bone-Marrow Transplant Program of Hematology Service at Reina Sofía University Hospital (Córdoba, Spain), after signed written informed-consent. MSC were cultured in Minimum Essential-Medium Alpha (MEM α) Eagle from Lonza (Basel, Switzerland), supplemented with 10% fetal bovine-serum (FBS) from Gibco - Life Technologies - Thermo Fisher Scientific (Waltham, MA, USA), 2 mM UltraGlutamine (Lonza), 1 ng basic fibroblast-growth factor (bFGF)/ml from Sigma-Aldrich (Saint Louis, MO, USA), 100 U ampicillin and 0.1 mg streptomycin/ml. Cells were grown at 37 ºC and 5% CO2 in humidity. Cultures grown until about 80 to 90% confluence were maintained in MEMα plus 10% FBS without bFGF (controls, C) or likewise but supplemented with 5x10–7 M dexamethasone, 0.5 mM isobutylmethylxanthine and 50 mM indomethacin (Sigma-Aldrich) adipogenic inductors for cell differentiation, in absence (A) or presence (A+O) 10–5 M oleuropein (purity >80%) from Extrasynthese (Lyon, France). Adipogenic differentiation at day 14 was determined by oil-red O staining of lipid droplets. In short, cells were fixed with 3.7% formaldehyde, washed with 60% isopropanol in water and stained with a mixture of 60% of 0.3% (w/v in isopropanol) of oil-red O and 40% distilled water for 20 minutes. They were then washed with water, stained with hematoxylin (all chemicals from Sigma-Aldrich) and visualized under a light microscope. Total RNA was isolated at days seven and 14 after adipogenesis induction, using mirVana miRNA Isolation Kit from Ambion - Life Technologies - Thermo Fisher Scientific. RNA were quantified using a ND-1000 spectrophotometer from NanoDrop - Thermo Fisher Scientific. Integrity was checked by agarose-gel electrophoresis (AGE). RNA samples were pooled from each time and donor, to generate three  samples: control (C), adipocytes (A) and adipocytes plus oleuropein (A+O) for gene-expression analyses.
attributes.tag: Experimental Factor: none
attributes.value: compound