2.9 RT-qPCR
Cells were seeded onto each scaffold or cultured as a monolayer (as described above) in triplicate and cultivated for 7 days in GM only, or for 7 days in GM followed by an additional 7 days in differentiation medium (DM: high glucose DMEM supplemented with 2% horse serum [Gibco] and 1% anti-anti [Gibco]). Both GM and DM were replaced with fresh medium every two days. All cells from the triplicate were then harvested in 1 mL TriReagent (Sigma-Aldrich) and the total RNA was isolated according to the manufacturer’s instructions. Next, 1 μg of each total RNA sample was converted into cDNA, following the instructions in the RevertAid H minus first strand cDNA synthesis kit (Thermo Fischer Scientific). RT-qPCR was performed using a Corbett 3000 device (Qiagen, Helden, Germany), 0.4–0.8 μM of each primer, 1 μl (diluted 1:10) of each cDNA, and 5 μl of iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, USA) in a final volume of 10 μl. Reactions were performed as follows: 50 °C for 2 min, 95 °C for 2 min, followed by 45 cycles of 94 °C for 15 sec, 60–62 °C for 15 sec, and 72 ° for 20 sec. The dissociation step was performed at the end of the amplification step to identify the specific melting temperature for each primer set.
GAPDH was used as a reference gene. Relative gene expression was determined using REST2009 software (based on the model by Pfaffl et al., 2001) [28]. MyoD , MyoG and Myf5 were used as target genes with the following primers: for MyoD(GTGGCAGCGAGCACTACA and GACACAGCCGCACTCTTC), for MyoG(TGAGAGAGAAGGGGGAGGAG and CGGTATCATCAGCACAGGAG), and for Myf5(GCAAAGACCCGTGACTTCAC and GCATGTGGAAAAGTGATA). The relative gene expression for each gene was determined by comparing the levels of gene expression in cells cultivated onto scaffolds and in cells cultivated in a monolayer. REST 2009 software [28] was used to determine statistical significance.