Ambion s magmax 96 viral rna isolation kit

For antemortem samples (probangs), rRT-PCR was performed as described below without any other treatment. For postmortem samples (tissues), two specimens of each tissue listed in Table 2 were thawed and immediately macerated in a TissueLyser bead beater (Qiagen, Valencia, CA) as previously described [35 (link)]. For RNA extraction of probang samples and macerated tissues, 50 μl of each sample was transferred to a 96-well plate (Thermo Scientific, Waltham, MA) containing 150 μl lysis/binding solution. RNA was subsequently extracted using Ambion’s MagMax-96 Viral RNA Isolation Kit (Ambion, Austin, TX) on a King Fisher-96 Magnetic Particle Processor (Thermo Scientific, Waltham, MA). RNA was eluted in a final volume of 25 μl. Once extracted, 2.5 μl of RNA was analyzed by rRT-PCR on the ABI 7000 system (Applied Biosystems, Austin, TX) as previously described [35 (link)]. Samples with cycle threshold (Ct) values < 40 were considered positive. rRT-PCR results were converted to FMDV RNA copy numbers per mg of tissue as previously described [36 (link)]. The Ct positivity cutoff of 40 corresponded to a detection threshold value of 2.24 log₁₀ FMDV RNA copies/mg (FMDV RNA/mg) of tissue. Real-time rRT-PCR results reported in Table 2 are the higher FMDV RNA/mg value of the two samples processed per tissue per animal.

To contrast the viral growth dynamics during infection in pigs between rNJ0612NME6 and M51R, the presence of viral RNA extracted from different biological samples collected during the time course of the experiment was assessed by real-time RT-PCR (rRT-PCR). RNA extraction was conducted using the Ambion’s MagMax-96 Viral RNA Isolation Kit (Ambion, Austin, TX) as previously described (Arzt et al., 2010 (link)). rRT-PCR targeting the VSNJV nucleocapsid gene (N) was conducted as previously described (Scherer et al., 2007 (link)) but using a different forward primer (5′-GCACTTCCTGATGGGAAATCA-3′) to match the native sequence of the NJ0612NME6 viral strain (Velazquez-Salinas et al., 2018b (link)). rRT-PCR was carried out using 2.5 μl of RNA on an ABI 7500 system (Applied Biosystems, Austin, TX). Results were expressed as RNA genome copy numbers per 2.5 μl of RNA.