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.