Myoblasts were grown to confluence, allowed to differentiate for 6 days, and then preincubated for 24 h with different fatty acids (oleic acid [OA], palmitic acid [PA], eicosapentaenoic acid [EPA] or linoleic acid [LA], each at 100 µM) or bovine serum albumin [BSA] (40 µM) before RNA isolation.
Growth protocol
Satellite cells were isolated from the M. obliquus internus abdominis of 3 healthy female donors. 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 from the primary myotubes using Agilent Total RNA isolation kit according to the supplier’s protocol (Agilent Technologies, Santa Clara, CA, USA). RNA was used individually and 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
The Affymetrix GeneChip RNA One cycle Amplification Kit was used to prepare labelled cRNA from 5 ug of total RNA. The protocol was conducted using the reagents provided by Affymetrix, as per the manufacturer's instructions.
Hybridization protocol
Hybridisation of 10ug cRNA was done overnight for 16 hours at 45ºC in a Hybridisation Oven 640 (Affymetrix). The protocol is conducted as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Target Hybridization) (P/N 701027, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described n 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).
