Tissuelyser lt device

Cells were lysed with RNA Lysis Buffer (PeqGOLD Total RNA kit, PeqLab), or TRIzol LS (Thermo Fisher Scientific), and kept at − 80 °C until total mRNA extraction, according to the suppliers instructions. Fetal liver (FL) pieces from wildtype slaughtered pig fetuses at day 25 of gestation, provided by FLI/Mariensee, were immediately flash frozen in liquid nitrogen and kept at − 80 °C until processing. FL pieces were first homogenized with TRIzol LS solution using stainless steel beads and processed by a TissueLyser LT device (Qiagen). The resultant homogenized solution was used for total mRNA extraction according to the TRIzol LS instructions protocol. Total mRNA from all samples was sub-sequentially treated with TURBO DNA-free Kit (Thermo Fisher Scientific), for complete removal of genomic DNA. The final product was quantified using the spectrophotometer Nanodrop N-1000 and processed for single stranded cDNA synthesis from 1 ug total mRNA in a final volume reaction of 10 uL using the High Capacity cDNA synthesis Kit (Thermo Fisher Scientific). The final cDNA was diluted 1:10 with RNAse and DNAse-free H₂O and used for qRT-PCR using the StepOnePlus device (AppliedBiosystems). All TaqMan probes used in this study are listed in Supplementary Table S3.

Total RNA was extracted with TRIZOL reagent (ThermoFisher), by homogenizing snap frozen tissue using a TissueLyser LT device (Qiagen). RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) as per manufacturer’s instructions. Quantitative RT-PCR (RT-qPCR) reactions were performed by means of the SYBR green method, using either the 2 × Master mix qPCR Low Rox kit (PCR Biosystems). The 2^−ΔΔCt method was used, normalizing to actin expression. Reverse transcriptase-minus and blank reactions were included in all experiments. Primers used were as follows: Spry1 Fwd 5´-CTCTGCGGGCTAAGGAGC-3´; Spry1 Rev 5´-ACGCCGGCTGATCTTGC-3´; Actin Fwd 5´-TTCTTTGCAGCTCCTTCGTT-3´; Actin Rev 5´-ATGGAGGGGAATACAGCCC-3´.

For the initial diagnostic assessment, nucleic acid from all samples had been extracted using the QiaAMP DNA Stool Mini Kit (Qiagen, Hilden, Germany) as described by the manufacturer without particular focus on specialized procedures for parasite DNA (later referred to as “without bead beating”). If any helminth real-time PCR signal had been detectable in the initial diagnostic process and residual stool material was still available stored at −80 °C, nucleic acid from these residual materials were extracted using polyvinylpyrrolidone pretreatment, bead-beating using garnet beads (0.7–1.2 mm, Biolabproducts, Bebensee, Germany) with a TissueLyser LT device (Qiagen) for 3 min at 30 s⁻¹, proteinase K digestion, and QiaAMP spin column extraction (Qiagen) as described previously [33 (link)]. This procedure is later referred to as “with bead beating”. Identical elution volumes as in the QiaAMP DNA Stool Mini Kit-based approach were achieved. To ensure sufficient sample volumes for the real-time PCR assays, photometric assessment of DNA concentrations within the samples using a Pico 100 Picodrop Microliter Spectrophotometer (Picodrop Ltd., Hinxton, UK) according to the manufacturer’s instructions was only performed from remaining residual DNA samples after the real-time PCRs had been completed.