An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV

Peña, Andrea A; Bols, Niels C; Marshall, Sergio H

qRT-PCR was carried out using a MJ Research realtime cycler. Each reaction for amplification of housekeeping candidates contained: 10 μl of the Brilliant II SYBR Green qPCR Master Mix (Stratagene), 100 nM of forward and reverse primers and 2 μl of 10-fold diluted cDNA, to a final volume of 20 μl. PCR was achieved with 10 min activation and denaturation step at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at the specific annealing temperature (see Additional file 1), 30 s at 72°C and 2 s at 74°C for fluorescence measurement. Following the final cycle, melting curve analysis were performed to examine the specificity in each reaction tube (absence of primer dimers and other non-specific products) by heating the samples from 60 to 90°C in 0.2°C increments with a dwell time at each temperature of 5 s while continuously monitoring the fluorescence.