Qiasymphony rna robotic system

RNA was extracted from 5 mg organ samples (gill, heart, anterior kidney) using the QIAsymphony® RNA robotic system (Qiagen) according to the manufacturers’ protocol and eluted in 100 μL RNase-free dH₂O. The RNA was reverse transcribed to cDNA using the TaqMan® Reverse Transcription Reagents kit (Life Technologies, UK) with oligo-d(T)₁₆ as described previously [33 (link)] using a 20 μL total reaction volume. Real-time RT-PCR assays were performed as described in McBeath et al. [37 ]. Primer and probe sequences (Additional file 1) for the assays targeting viral gene (no differentiation of ORFs in either assay) segments 7 (seg7) and 8 (seg8) from both European and North-American genogroups, immune markers Type I (α1 and α2), Type II interferon (IFN), Mx, γIFN-induced protein (γIP) and endogenous control elongation factor 1α (ELF), have been described previously [21 (link),33 (link)]. Absolute quantitation of transcripts was carried out. The cycle crossing point (Cp) values were converted into expression values normalised against the reference gene, ELF, using the statistical standard curve method to produce relative expression ratios [39 (link)].

Total RNA was extracted using either the QIAsymphony^® RNA robotic system (Qiagen) (challenge material; 5 mg hind gut and gill, 20 mg pectoral fin and skin/mucus swabs) or the RNeasy tissue kit (Qiagen) (ISA outbreak samples; 5 mg) according to manufacturer’s protocols. For hind gut and all ISA outbreak samples, cDNA was synthesized using the TaqMan^® Reverse Transcription Reagents kit (Life Technologies, UK) with oligo-d(T)₁₆ as described previously [21 (link)] and real-time PCR (qPCR) analysis was performed on a Lightcycler LC96 (Roche) as described in [6 (link)] using assays targeting ELF and ISAV segment 8 [7 (link)]. For skin swabs and pectoral fin, one-step real-time RT-qPCR (Quantitect Probe RT-PCR kit, Qiagen) was performed as outlined in McBeath et al. [7 (link)]. Transcription of immune genes type I and II IFN, Mx and γIP were analysed on a Lightcycler LC96 (Roche) for all tissues and statistical analysis was performed as previously described [7 (link)]. RNA species specific RT and qPCR to specifically examine replication (cRNA) and transcription (mRNA) was performed according to McBeath et al. [7 (link), 22 (link)].