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

Total RNA extraction from cell cultures was carried out using Trizol® (Invitrogen) according to the manufacturer instructions. A NanoDrop ND 1000 spectrophotometer was employed to analyze RNA concentration and purity. All samples were DNase treated (RQ1 RNase-free DNase, Promega) to remove any contaminating DNA. For PCR amplification, first strand cDNA was synthesized from 1 μg total RNA using oligo(dT) primer and the AffinityScript™ QPCR cDNA Synthesis Kit (Stratagene). CHSE-214 and RTS11 cultures were monitored for infection by phase contrast microscopy (Olympus IMT-2 microscope). P. salmonis PCR confirmation was carried out by using the primer pair RTS1/RTS4 against the ITS region of the bacterial 16S rRNA operon as described previously [22] . IPNV infections were confirmed by using 1 step RT-PCR procedure (Brilliant QRT-PCR Master Mix Kit 1-Step, Stratagene) with primer set VP2SNP-F/VP2SNP-R (Santi, unpublished) against to the VP2 fragment sequence. Reverse transcription was performed by incubating at 50°C for 55 min followed by PCR amplification (95°C for 10 min, 35 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C, and 72°C for 10 min). Mycoplasma contamination was absent as tested by amplifying with the primer set MyF1/MyR1 (PCR Mycoplasma Detection Set -TaKaRa Biomedicals Takara Shuzo).