Qubit fluorometer v2

DNA was extracted from 5 to 10 10 μm thick scrolls of formalin-fixed-paraffin-embedded (FFPE) tissue using the MasterPure Complete DNA and RNA Purification Kit (Epicentre, WI, USA) according to the manufacturer’s instructions with a modified proteinase K digestion in which incubation time was increased from 24 to 48 h. Total RNA was extracted from FFPE tissue with the RNeasy FFPE extraction kit (QIAGEN, CA, USA) using the manufacturer’s guidelines. RNA/DNA quality was assessed based on 260/280 values obtained using the NanoDrop 2000 (Thermo Scientific, DE, USA) and samples between 1.7–1.9 and 1.9–2.0 were considered to be of sufficient quality for DNA and RNA, respectively. Samples were quantified using the Qubit Fluorometer V2.0 (Thermo Scientific, DE, USA).

DNA was extracted from 3–5 10 µm thick scrolls obtained from formalin-fixed paraffin embedded (FFPE) tissue either shaved from the original block or scraped from unstained slides. The extraction was completed with the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). If available, 10–20 mg of fresh frozen tissue rather than FFPE was used for extraction with the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). DNA was quantified with the Qubit Fluorometer V2.0 using the dsDNA Broad Range Assay Kit (Thermo Scientific, Waltham, MA). All assay kits and quantification methods were used according to the manufacturer’s guidelines.
RNA was extracted from 3 to 5 10 µm thick scrolls obtained from FFPE either shaved from the original block or scraped from unstained slides. Extraction was completed using the ExpressArt FFPE Clear RNA extraction kit (Amsbio, Cambridge, MA). If available, 10–20 mg of fresh frozen tissue rather than FFPE was used for extraction using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA was quantified with the Qubit Fluorometer V2.0 using the RNA Broad Range Assay Kit (Thermo Scientific, Waltham, MA). All assay kits and quantification methods were used according to the manufacturer’s guidelines.

Genomic DNA was extracted from 50 mg of faecal pellets using the DNeasy Blood and Tissue kit as per manufacturer’s instructions and described in detail at https://www.protocols.io/view/genomic-dna-extraction-from-animal-faecal-tissues-6bbhain (Qiagen, Chadstone Centre, Victoria, UK). Hare and rabbit samples were processed separately and reagent-only controls (ROC) were included with each set of extractions. All samples were quantified and evaluated for integrity using the Qubit dsDNA Broad Range assay kit and the Qubit Fluorometer v2.0 (Thermo Fisher Scientific, Waltham, MA, USA) and Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA samples were stored at −20 °C.