**2.2.3 qPCR amplification**

qPCR reactions were conducted with the primer sets indicated on table 1. Quantification of transcripts were analysed by qPCR with SYBR Green chemistry (SYBR Green I Master, Roche) on a LightCycler® 480 (Roche) as previously described (Campos et al., 2010). Fiftyfold diluted muscle cDNA were run in duplicate, and minus reverse transcriptase and no template controls were included in the reactions. Thermocycling parameters were as follows: 95°C for 15 min, followed by 45 cycles of 15 s at 94°C, 20 s at 60°C and 20 s at 72°C. Five-point standard curves of a 2-fold dilution series (1:1, 1:2, 1:4, 1:8 and 1:16) were prepared from pooled RNA that was reverse transcribed as above. These dilution curves were used to calculate amplification efficiencies of the PCR reactions (Fernandes et al., 2006). Cycle threshold (Ct) values were determined by the LightCycler® 480 software with a fluorescence level arbitrarily set to 1.


Table 1. Primers used in this study. Primer sequences, Genbank accession numbers, amplicon sizes and PCR efficiencies are indicated.

### **2.3 Data analyses**

#### **2.3.1 Expression stability analyses**

Raw qPCR data were converted to expression level using the above dilution curves. These were then analysed for expression stability using the statistical applications GeNorm (Vandesompele et al., 2002) and NormFinder (Andersen et al., 2004).
