Invitrogen purelink viral rna dna mini kit

We extracted RNA from 20–30 mg of liver tissue or bile using the RNeasy Mini Kit (QIAGEN, Hilden, Germany); the Maxwell 16 LEV simplyRNA Tissue Kit (Promega, Madison, WI, USA); or the Invitrogen Purelink viral RNA/DNA Mini Kit (ThermoFisher Scientific) following the manufacturers’ instructions. We performed first-strand cDNA synthesis from 5 μL of RNA with 500 ng of OligodT (18mer) (Geneworks, Thebarton, South Australia, Australia) using Invitrogen Superscript III (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s directions. We conducted initial detection and typing of RHDV samples submitted to CSIRO (>1 isolate from each outbreak) using the universal lagovirus RT-PCR, as described previously (35 (link)), followed by Sanger sequencing of amplified products. RHDV G2 genomes were amplified as described previously (7 (link)). We amplified overlapping fragments of the RHDVa genome using Platinum Taq DNA polymerase high fidelity (Life Technologies) using primer sets detailed in Table 2 (cycling conditions available from the authors upon request). We purified PCR products using the QIAquick PCR purification kit (QIAGEN), quantified with the Qubit dsDNA BR assay (Life Technologies) and pooled in equimolar ratios.