2.6. mRNA expression levels

cDNA was synthesized using a Tetro cDNA Synthesis kit (Bioline #BIO-65026 Luckenwalde, Germany) in accordance with the manufactures’ instructions. Briefly, the reaction conditions were as follows: 1 μg of RNA, 1 μl of random primers, 1 μl of 10 mM dNTP, 1 μl of RNase inhibitor, and 0.25 μl of 200 U/μl reverse transcriptase in a final volume of 20 μl. The solution was incubated for 10 min at 25 °C for primer annealing, followed by 42 °C for 60 min for primer elongation, and followed by 80 °C for 5 min termination. cDNA samples were stored at − 20 °C.

Based on the principle of the SybrGreen detection method, EvaGreen® dye (Biotium, Hayward, CA, USA) was used to detect PCR products. The PCR was performed using a primer pair specific for mRNA of vascular endothelial growth factor (VEGF), silent mating type information regulation 2 homolog 1 (SIRT1), forkhead box protein O1 (FOXO1), mitochondrial calcium uniporter (MCU, Insulin like growth factor (IGF-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and mechano growth factors (MGF) isoforms (for sequences of mRNA genes used in the study see Table 1). PCR amplifications consisted of equal amounts of template DNA, 10 μl of ImmoMix™ complete ready-to-use heat-activated 2× reaction mix (Bioline GmbH, Luckenwalde, Germany), 1 μl of 20x EvaGreen (Biotium, Hayward, CA, USA), 2.5 μl of 10 nmol/L forward and reverse primer (IBAGmbH, Göttingen, Germany) and water to a final volume of 20 μl. Amplifications were performed in a Rotor-Gene 6000 thermal cycler (Corbett Life Science/Qiagen, London, UK) at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s in triplicates. The validity of the signal was evaluated by melting analysis and agarose gel electrophoresis. Human 28 S rRNA gene served as an endogenous control gene (Table 1).

Reference genes.