Nucleospin rna virus extraction kit

Supernatants from infected cell cultures were centrifuged at 1,500 g for 10 min at 4°C to pellet any cellular debris, and clarified by centrifuging twice at 10,000g for 30 min at 4°C. Viruses were concentrated by ultracentrifugation at 100,000g for 1 h at 4°C. The pellets were resuspended in 1 ml of PBS and loaded onto pre-formed 6–50% iodixanol-(OptiPrep, Axis-Shield) sucrose step gradients, which were centrifuged at 205,000g for 2 h and 45 min at 4°C (SW41 Ti rotor in a Beckman Coulter Optima L-90K centrifuge). Approximately 20 fractions of 0.5 ml each were collected from the gradients and the density of each fraction was determined using a refractometer.
RNA from gradient fractions was extracted using the Nucleospin RNA Virus Extraction Kit (Macherey-Nagel) and HAV genome copy numbers were determined by a previously described Real-Time RT-PCR of the 5’ non-coding region (Costafreda et al., 2006 (link)), using the RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen).

The PRRSV RNA in serum was evaluated by quantitative PCR (qPCR) after PRRSV challenge. The primers specific for the ORF5 gene of either PRRSV-1 or PRRSV-2 and detection conditions were described previously^{12 (link)}. In brief, total RNA was extracted using NucleoSpin RNA Virus extraction kit (Macherey-Nagel, Duren, Germany) in accordance with manufacturer’s instructions. The quality of RNA was measured using spectrophotometer (Colibri, Titertek-Berthold, Pforzheim, Germany), and converted to cDNA. All cDNA was used for quantitative PCR (qPCR). PRRSV RNA was quantified using ABI PRISM 7500 Real time PCR platform (Applied Biosystem, CA, USA). Primers specific for the ORF5 gene of either PRRSV-1 or PRRSV 2 were used. Each qPCR reaction contained 0.1 μg of cDNA, 0.2 μM of each primers, 1x Eva Green real-time-PCR master mix E4 (GeneOn GmbH, Ludwigshafen, Germany), and deionized water to yield a 20 ul final volume. The thermal profile for qPCR was 94 °C for 4 min, followed by 35 cycles of 94 °C for 45 s, 55 °C for 45 s, and fluorescence acquisition at 72 °C for 45 s. pGEM-T Easy Vector (Promega, WI, USA) containing an inserted ORF5 gene of each PRRSV was used to construct plasmid standards. A standard curve was generated using serial diluted plasmid standards of 10⁰–10⁷ copies/μl. Copy number of the PRRSV RNA was calculated using standard curve method.

Three disks of 2 mm diameter were punched out of each FTA^® card’s circle using a Whatman Harris Uni-Core^™ puncher and pooled together in a 2 mL microcentrifuge tube with 300 µL of TE Buffer (10 mM Tris, 1 mM EDTA, pH8.0) to soak the disks. The puncher was cleaned using pure ethanol between circles and cards. For nucleic acid elution, microcentrifuge tubes were vortexed for two hours with Vortex-Genie^® 2 then briefly centrifuged. Then 150 µL of eluent were used for RNA and DNA extraction using the NucleoSpin RNA Virus extraction kit (Macherey–Nagel, Düren, Germany) following the manufacturer’s instructions.