Rnaase free water

Cell extracts were obtained by cell lysis using NucleoZOL (Mecherey-Nagel, Dueren, Germany). To further characterize the molecular phenotype of cardiomyocyte-like cells during their differentiation, cells were harvested and lysed on days 0, 3, and 5 of their differentiation. Stretched cardiomyotubes were harvested 12 h after the completion of the stretching protocol, while control (nonstretched) myotubes were also harvested 12 h after the end of each protocol. Total RNA was isolated from the lysates according to the manufacturer’s recommendations. The extracted RNA was dissolved in RNAase-free water (Invitrogen, Waltham, MA, USA) and the concentration and purity were determined spectrophotometrically (Thermo Nanodrop 2000,Thermo Scientific, Waltham, MA, USA) by absorption at 260 and 280 nm. Integrity of total RNA was confirmed by visual inspection of the electrophoretic pattern of 18S and 28S ribosomal RNA in ethidium bromide-stained 1% agarose gels under ultraviolet (UV) light. The total RNA samples were stored at −80 °C until further analysis for the determination of the mRNA levels of the genes of interest by reverse transcription and semi-quantitative real-time polymerase chain reaction (PCR) procedures.

Both control and aged C2C12 cells were harvested at the 0, 2nd, and 6th day of differentiation, and cell extracts were obtained by cell lysis using NucleoZOL (Mecherey-Nagel, Duren, Germany). Total RNA was isolated from the lysates according to the manufacturer’s recommendations. The extracted RNA was dissolved in RNAase-free water (Invitrogen, Carlsbad, CA, USA) and the concentration and purity were determined spectrophotometrically (ThermoNanodrop 2000, Thermo Scientific™, Waltham, MA, USA) by absorption at 260 and 280 nm. The integrity of total RNA was confirmed by visual inspection of the electrophoretic pattern of 18S and 28S ribosomal RNA in ethidium bromide-stained 1% agarose gels under ultraviolet (UV) light. The total RNA samples were stored at –80 °C until further analyses for the determination of the mRNA levels of the genes of interest by reverse transcription and semi-quantitative real-time polymerase chain reaction (PCR) procedures.