Endangered wild salmon infected by newly discovered viruses

Mordecai, Gideon J; Miller, Kristina M; Di Cicco, Emiliano; Schulze, Angela D; Kaukinen, Karia H; Ming, Tobi J; Li, Shaorong; Tabata, Amy; Teffer, Amy; Patterson, David A; Ferguson, Hugh W; Suttle, Curtis A

Chinook smolt samples positive for PsNV from 2014 were used for tissue localization ( Figure 4A ). Gill, liver, heart, kidney, and brain were individually homogenized, processed for RNA extraction (as described above), and 1 ug normalised RNA was used for reverse transcription. Resultant cDNA for each individual tissue was used as the template for PsNV relative quantification using an ABI 7900HT (ABI) in 384-well optical plates. The qPCR reaction volume was 12 ml, which comprised 6 ml of 2X TaqMan Gene Expression Master Mix (ABI PN 4369016), 4.3 ml of water, 0.22 ml of mixed forward and reverse primers (900 nM final concentration of each), 0.24 ml of each probe (200 nM final concentration; assay specific probe and APC control probe), and 1 ml of cDNA template. Temperature cycles included one 2 min hold (50˚C), a 10 min denaturation (95˚C), and 40 cycles of denaturation (95˚C for 15 s), annealing and extension (60˚C for 60 s). Amplification conditions on the ABI 7900 were not optimised for this platform, but rather closely reflected those used on the BioMark platform. Samples run on the ABI did not undergo STA enrichment. Standard curves were constructed using the same APC clone standards spiked in with CHSE DNA as on the BioMark. Serial dilutions were made to obtain concentrations of 24, 1.2 Â 102, 6 Â 102, 3 Â 103, 1.5 Â 104, 1.5 Â 105 copies of the clone per reaction. Clone standards, unknown samples, positive and negative controls were all run in duplicate. The ABI software calculates the relative copy number based upon the serial dilution of the standard curve.