Magmax express magnetic particle processor

Total RNA was extracted from the lysed, restimulated anterior kidney leukocytes using a MagMAX™-96 Total RNA Isolation Kit (Life Technologies, USA) in an automated setup on a MagMax™ Express Magnetic Particle Processor (Applied Biosystems, USA) according to the manufacturer’s instructions. Subsequently, the samples were tested for quantity and purity on a NanoDrop™ 2000 (Thermo Scientific) and were stored at −80 °C until further processing. cDNA synthesis was carried out using the TaqMan^® Reverse Transcription Reagents kit (Applied Biosystems), using DNase/RNase-free UltraPure™ Distilled Water (Invitrogen, Denmark). An Oligo d(T)₁₆-primer included in the kit was used and the cDNA synthesis itself was carried out in µLtraAmp PCR plates (Sorenson BioScience, Inc., Salt Lake City, USA) with MicroAMP^® cap-strips on a T100™ Thermal Cycler (Bio-Rad, Copenhagen, Denmark) 25 °C, 10 min -> 37 °C, 60 min -> 95 °C, 5 min).

SwIV titer in BAL fluid collected at DPC 7 from euthanized pigs was quantified by qRT-PCR as previously described [28 (link)]. Briefly, total RNA was extracted from 50 μl of BAL fluid using MagMAX™-96 RNA Multi-Sample Kit (Ambion) according to the protocol provided by the manufacturer. The automated MagMAX™ Express Magnetic Particle Processor (Life Technology) was used for sample processing, and qPCR was performed to estimate viral RNA load in samples. SwIV RNA load in test samples were calculated using the standard curve with Y-axis being virus concentration in TCID₅₀ and X-axis was being the Ct value.
The replicating challenged SwIV in the BAL fluid was quantified by the indirect immunofluorescence assay (IFA) as described previously [15 (link)]. Briefly, MDCK cell monolayer cultured in 96-well plate was treated with ten-fold serially diluted BAL fluid (100 μl) diluted in serum free DMEM and supplemented with TPCK trypsin (1 μg/mL), and after 24 hr cells were washed and fixed in 80% acetone in water. The air dried plates were immunostained using the influenza nucleoprotein specific mAb (M058, CalBioreagents, CA) followed by goat anti-mouse IgG Alexa 488 conjugated secondary antibody (Invitrogen). The plates were washed and treated with PBS/Glycerol (60:40) and the immunofluorescence was evaluated using an inverted fluorescent microscope (Olympus IX51, Center Valley, PA).

Fifty microlitre of supernatant from processed clinical specimens or following cell culture passage was used for nucleotide extraction using the MagMax 96 Viral RNA Kit (ThermoFisher Scientific) in a MagMAX Express Magnetic Particle Processor (ThermoFisher Scientific) following manufacturer’s instructions. Extracted RNA was used for PCR testing immediately or stored at −80 °C for further use.

Total RNA was extracted using an MagMAX™ Express Magnetic Particle Processor (Thermo Fisher Scientific) using the MagMAX™ Total Nucleic Acid Isolation Kit (Applied Biosystems part number AM1830). Extractions were performed according to the manufacturer’s instructions with minor changes. In brief, 2000 µL detached cell cultures were centrifuged for 5 min at 8000 g to collect the pellet. Supernatants were discarded and 300 µL Millipore ultra-pure water (18.2 Ω) was added to break the cells by means of osmosis. A total of 50 µL of sample homogenate were added to the Bead Mix≈in the row A of the 96-well processing plate wells along with 65 µL of lysis solution and 65 µL isopropanol. In rows B and C, 150 µL of Wash Solution 1 was added while Wash Solution 2 was added to rows D and E. Row F was filled with 50 µL of Elution Buffer. The extraction programme was according to the manufacturer’s instruction. Briefly, lysis binding was conducted for 5 min followed by washing with each wash solution for 30 s. The RNA was then air-dried and re-suspended in Elution Buffer.
Real-time reverse transcriptase quantitative PCR, targeting segment 10, was performed as described by Steyn et al. (2015 ).