Quantifluor one system

Total DNA was extracted from GTE, EGCG, or CTRL rat left ventricles (LVs) by using the commercial Kit NucleoSpin Tissue (Macherey-Nagel, Duren, Germany) according to manufacturer’s instructions and quantified by a Biospectrophotometer (Eppendorf AG, Germany). Then, 0.1 ng of DNA, quantified by the QuantiFluor ONE System using the Quantus Fluorometer (Promega Corporation, Madison, USA), was used for qPCR amplification by the SSO-Advanced Universal SYBR Green Supermix (Bio-Rad). Mitochondrial DNA (mito gene) corresponding to sequences of the NADH-ubiquinone oxidoreductase subunit 1 (ND1) and the NADH-ubiquinone oxidoreductase subunit 3 (ND3) was amplified using the primer set shown in Table 2. Primers were carefully chosen in order to avoid amplification of nuclear mitochondrial insertion sequences (pseudogenes) that might negatively affect accurate mtDNA quantification. The GAPDH gene was used as a nuclear DNA marker (nucl gene). Thermal cycler conditions consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 20 s. To calculate the relative mtDNA content, the following equations were used:

DNA extraction for tissue samples was done using the DNeasy Blood & Tissue Kit (Qiagen) following the protocol for purification of total DNA from animal tissues (Spin‐Column Protocol, Qiagen). Pieces of 25 mg consisting of muscle, tendon, bone, or some hair were cut off from the samples and stored in 2 ml tubes at −20°C until further analysis. DNA concentration was assessed using quantus (QuantiFluor ONE System, Promega). The quality of the DNA samples was estimated with nanodrop 2000 (Thermo Fisher Scientific), based on the 260/280 nm ratio and concentration of the DNA, and quantitatively by the spectrum observed. Additionally, DNA quality was checked for all samples through electrophoresis using EZ‐Vision Blue Light Dye (Amresco, LLC) on a 1% agarose gel.
Based on the concentrations assessed using quantus (Promega), DNA samples were diluted to 2.5 ng/μl. Genotyping of tissue samples followed the protocol described above for NiG samples.