Comprehensive RNA extraction and analysis for disease detection

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)].