An aliquot of 0.5 μg total RNA was treated with 1 unit DNAse (Fermentas, St. Leon-Rot, Germany) 30 min at 37°C. Reverse transcription of RNA (0.5 μg) was performed with oligo (dT)12–18 primer and 200 units of SUPERSCRIPT II (Invitrogen, Karlsruhe, Germany) and 24 units of Ribo LockTM RNAse inhibitor (Fermentas) for 1 h at 42°C. The cDNA was used for PCR analysis. All cDNA probes were analyzed for: ACTA1, (NM_001100), amplicon length 85 bp; MYOG, (NM_002479), amplicon length 113 bp; MYH3, (NM_002470), amplicon length 84 bp and the RG: ACTB (NM_001101), amplicon length 104 bp; B2M, (NM_004048), amplicon length 98 bp; GAPDH, (NM_002046), amplicon length 119 bp; cyclophilin A/PPIA, (NM_203430), amplicon length 121 bp; RPLPO, (NM_001002), amplicon length 170 bp; TBP, (NM_003194), amplicon length 132 bp. The QuantiTect/PrimerAssays were purchased from QIAGEN GmbH (Hilden, Germany). cDNAs were amplified with Brilliant® II SYBR® Green QRT-PCR Master Mix (Stratagene-Agilent Technologies, Waldbronn, Germany). The thermal profile consisted of 1 cycle at 50°C for 2 minutes followed by 1 cycle at 95°C (2 min), 45 cycles at 95°C (15 sec), 60°C (1 min). Amplification was performed using the Mx3005P™ QPCR System (Stratagene). For relative quantification, a standard curve was generated in every individual run. Shortly, total RNA was pooled from muscle biopsies of healthy human volunteers, reverse transcription was performed and a serial dilution of the cDNA was used to perform the calibration curve. The data were analyzed using the relative standard curve method. For each unknown sample, the relative amount is calculated using linear regression analysis from their respective standard curves. Data were analyzed using the Mx3005P analysis software (Stratagene-Agilent Technologies, Waldbronn, Germany). The efficiencies of all GOI and RG were calculated in every individual run (Table 1 ).
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