Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

Olsvik, Pål A; Lie, Kai K; Jordal, Ann-Elise O; Nilsen, Tom O; Hordvik, Ivar

RNA was isolated with phenol-chloroform extraction as described by Chomczynski and Sacchi [28] , and stored in 100 µl RNase-free MilliQ H 2 O. Total RNA was extracted using Trizol reagent (Invitrogen, Life Technologies), according to the manufacturer's instructions. Genomic DNA was eliminated from the samples by DNase treatment according to the manufacturer's description (Ambion). The RNA was then stored at -80°C before further processing. The quality of the RNA was assessed with the NanoDrop ® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A 260/280 nm absorbance ratio of 1.8 -2.0 indicates a pure RNA sample. The RNA 6000 Nano LabChip ® kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the integrity of the RNA. We used the RNeasy MinElute Cleanup kit from Qiagen to purify our most troublesome samples. With this kit the A260/230 ratio increased on average by 5 % (n = 10).