Quantifying prdm1 Gene Expression by qPCR

To evaluate the levels of transcription of different prdm1 genes, real-time PCR was performed in a LightCycler 96 System instrument (Roche) using FastStart Essential DNA Green Master reagents (Roche) and specific primers designed using the mRNA sequence from each prdm1 gene (Table 1) and the Primer3 software (41 ). Each sample was subjected, in duplicate, to an initial cycle of denaturation (95°C for 10 min), followed by 40 amplification cycles (95°C for 10 s, 60°C for 10 s, and 72°C for 10 s). A dissociation curve was obtained by reading fluorescence every degree between 60 and 95°C to ensure only a single product was amplified. Negative controls with no template and minus reverse transcription controls (-RT) were included in all experiments. Gene expression was normalized to the relative expression of the rainbow trout elongation factor (EF-1α) amplified using primers previously used (42 (link), 43 (link)). Expression levels were calculated using the 2^-ΔCt method, where ΔCt is determined by subtracting the reference gene value from the target Ct as described previously (42 (link), 43 (link)).