Sds 7900 ht instrument

qRT-PCR was performed on RNA samples prepared as described above. The cDNA was produced by reverse-transcribing 400 ng of total RNA using a mix of random hexamers and oligo d(T) primers and Primescript reverse transcriptase enzyme (Takara Bio, San Jose, CA, USA). The efficiency of each pair of primers was tested with serial dilutions of cDNA. Oligonucleotides are indicated in Table 2. PCR reactions (10 µL volume) contained 1:20 diluted cDNA, 2 × Power SYBR Green Master Mix (Applied Biosystems by ThermoFisher, Waltham, MA, USA), and 300 nM of forward and reverse primers. PCRs were performed on a SDS 7900 HT instrument (Applied Biosystems by ThermoFisher, Waltham, MA, USA) with the following parameters: 50 °C for two minutes, 95 °C for ten minutes, and 45 cycles of 95 °C 15 s, 60 °C one minute. Each reaction was performed in three replicates on 384-well plate. Raw Ct values obtained with SDS 2.2 (Applied Biosystems by Thermo Fisher, Waltham, MA, USA) were imported into Excel and normalization factors were calculated using the GeNorm method as described by Vandesompele et al. [34 (link)].

RNA extraction (myoblasts, 48 h-MuRC and 96 h-MuRC) was performed with TRIzol reagent (Life Technologies) according to the manufacturer's protocol. After measuring sample concentrations, 0.5 µg of total RNA was reverse-transcribed with the PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer's instructions. qRT-PCR experiments were all performed at the iGE3 Genomics Platform of the University of Geneva (http://www.ige3.unige.ch/genomics-platform.php) on an SDS 7900 HT instrument (Applied Biosystems). Raw threshold-cycle (Ct) values obtained with SDS 2.2 (Applied Biosystems) were imported into Excel. Normalization factor were calculated using the geNorm method [34 (link)]. Primers used for qRT-PCR were: Hs β2-microglobulin forward, 5′-TGCTCGCGCTACTCTCTCTTT-3′; Hs β2-microglobulin reverse, 5′-TCTGCTGGATGACGTGAGTAAAC-3′; Hs EEF1A1 forward, 5′-AGCAAAAATGACCCACCAATG-3′; Hs EEF1A1 reverse, 5′-GGCCTGGATGGTTCAGGTA-3′; Hs Pax7 forward, 5′- AAACACAGCATCGACGGCA-3′; Hs Pax7 reverse, 5′- CTCGTCCAGCCGGTTCC-3′; Hs MyoD forward, 5′- TGCCACAACGGACGACTTC -3′; Hs MyoD reverse, 5′- CGGGTCCAGGTCTTCCGAA-3′; Hs HES1 forward, 5′- CAGATGACGGCTGCGCT -3′; Hs HES1 reverse, 5′- TCGGTACTTCCCCAGCACAC-3′.

Real-time PCR was used to measure the expression of p21, 14–3-3 (YWHAQ), GADD45, PUMA (BBC3), Bax, MDM2, Noxa (PMAIP1), CXCR4, and NMYC. GAPDH, EEF1A1, and TBP were used as a normalization control (Additional file 2: Table S1). PrimeScript Reverse Transcriptase enzyme (Takara Bio Inc., Japan) and 2x Power SYBR Green Master Mix (Applied Biosystems, USA) was used for generation of cDNA and real-time PCR according to the manufacturer’s recommendations. PCR was performed on an SDS 7900HT instrument (Applied Biosystems, USA). Raw Ct values obtained with SDS 2.2 (Applied Biosystems, USA) were imported in Excel and normalization factor and fold changes were calculated using the GeNorm method [30 (link)].