Tissuelyser 2 tissue homogenizer

Organ samples were homogenized in a serum-free medium using the TissueLyser II tissue homogenizer (Qiagen, Hilden, Germany). Genome extraction of samples taken during the animal trial and homogenized tissue samples was performed utilizing the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany) using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions with volume modifications as described previously [46 (link)]. To control successful DNA extraction and inhibition-free amplification, an internal control DNA (IC2-DNA) was added to the samples during the extraction process [47 (link),48 (link)]. Analyses of the presence of the capripox virus genome were performed using the pan capripox real-time qPCR of Bowden et al. [49 (link)] with a modified probe [50 (link)], utilizing the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA).

Efficient tumor tissue disruption and homogenization was performed with magnetic beads (5 mm stainless steel) in a TissueLyser II tissue homogenizer (Qiagen). For each tissue sample, one magnetic bead was introduced into the Eppendorf tube followed by 20 μl of ice-cold buffer per mg of tissue (MS-friendly extraction buffer: Urea 8 m/Tris 30 mm (pH 7.5–8) supplemented with PhosStop (Roche) and protease inhibitor (Roche, EDTA free)). Tissue samples were immediately disrupted (TissueLyser II parameters: 2.5 min at 20Hz, twice if needed), the beads removed, the samples briefly vortexed and gently mixed in a Thermomixer (Eppendorf Belgium, Rotselaar, Belgium) at 4 °C during 45 min. To further improve sample homogenization, two freeze-thaw cycles (−80 °C/ice-cold) and an ice-water bath sonication step (Bioruptor (Diagenode Europe, Seraing, Belgium) at medium sonication intensity (ON/OFF pulse time of 30 s for 5 min) were performed. Samples were centrifuged at 15,000 rcf (12,700 rpm) during 15 min (4 °C) to remove cell debris and unsolubilized components. Supernatants were transferred to fresh Eppendorf tubes and stored at −80 °C prior further treatment.

First, all organ samples were homogenized in serum-free medium using the TissueLyser II tissue homogenizer (QIAGEN, Hilden, Germany). DNA extraction from all samples taken during the animal trial (EDTA blood, serum, nasal swabs), as well, as the homogenized organ samples was performed with 100 mL sample material using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions on the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany). The extracted nucleic acid was eluated in 100 µL elution buffer. For control of successful DNA extraction, an internal control DNA (IC-2 DNA) was added during the extraction process [29 (link)]. Afterwards, a pan Capripox real-time qPCR using the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA) was performed according to the protocols of Bowden et al. (primer) [3 (link)] and Dietze et al. (probe) [30 (link)] using 2.5 µL template DNA in a 12.5 µL qPCR total volume.