μdrop plate

Post-mortem frozen human brain sections from MS and control donors were obtained from the Rocky Mountain MS Center Tissue Bank (Englewood, CO, USA) after approval by the Medical University of Gdansk (Poland) bioethics committee (NKBBN/253/2018). Lightly frozen 1 cm by 1 cm fragments were dissected from the white matter (WM) and periventricular WM plaques. Corresponding areas of WM were dissected from control brains. The fragments were deep-frozen in liquid nitrogen, ground using mortar and pestle, and homogenized in Fenozol reagent (A&A Biotechnology, 203–50). Total RNA was isolated from approximately 200 mg of homogenized tissue using the Total RNA Mini kit (A&A Biotechnology, 031–100) in an RNAase-free environment according to the manufacturer’s instructions. RNA quantification and quality control were performed spectrophotometrically at 260 nm and 280 nm using a PerkinElmer VICTOR Nivo plate reader (Perkin Elmer, USA) equipped with a μDrop Plate (Thermo Scientific, N12391). Isolated total RNA was stored at -20°C until used.

Leaf samples were collected from commercial macadamia trees with the permission of the owners at each private farm in Bundaberg and from the wild accessions in ex-situ germplasm conservation site in Queensland with the permission of the lead program manager (Professor Bruce Topp, The University of Queensland, National Macadamia Breeding and Conservation). We also collected leaf samples from the common ornamental tree B. celsissima, which is a close relative of macadamia and commercially available for ornamental purpose in Australia. All relevant institutional, national, and international guidelines, legislation and protocols were followed for the collection of the samples [26 ]. The identity of all plant materials was confirmed by O. A. Akinsanmi, The University of Queensland. Sampling methods used in this study are described in Zakeel et al. [27 ]. Sources and specimens for all plant materials used in this study are accessible in public collection in Australia. Total nucleic acids and pure RNA were extracted from the lamina and midrib of leaf samples using the CTAB method of Rogers and Bendich [28 ], and the TRIzol reagent as per the manufacturer’s instructions, respectively. RNA was quantified using a μDrop Plate (Thermo Scientific).

After treatment of HgCl₂ for 48 h, cells were harvest at 3000 rpm for 3 min. The solvent 0.6% EtOH treated cells were used as control. Total RNA was extracted using TRIzol. The amount of total RNA was determined using Thermo scientific Multiskan GO microplate reader with μDrop™ Plate (Waltham, MA). The quality of total RNA was evaluated using the Agilent 2100 Bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Santa Clara, CA, USA). The RNA used in RNA-seq was considered to be high based on a RIN value of 7.

On each sampling day, pools of spheroids were collected as previously detailed [22 (link)], centrifuged at 1500 rpm (239 RCF) for 5 min, and the pellets were snap-frozen in liquid nitrogen and stored at −80 °C. The total RNA extraction from spheroids and liver samples was carried out using an illustra^TM RNAspin Mini RNA isolation Kit (GE Healthcare, Chicago, IL, USA), according to the manufacturer’s recommendations. The protocol included a DNase I treatment step to avoid genomic DNA contamination of samples. RNA purity and quantification were checked in a Multiskan^TM GO microplate spectrophotometer (Thermo Scientific, Vantaa, Finland), using a μDrop™ Plate, with a SkanIt Microplate Reader software (Thermo Fischer Scientific). The λ 260/280 nm ratio was 2.2 ± 0.1 (mean ± standard deviation) for all samples, which is acceptable for a pure RNA sample (~2.0) [30 ]. Agarose gel with GelRed (Biotium, Fremont, CA, USA) staining allowed the RNA qualitative assessment. cDNA syntheses of spheroid and liver samples were made using an iScript™ Reverse Transcription Supermix kit (Bio-Rad, Hercules, CA, USA) for a total volume of 20 μL, using 300 ng of total RNA.

The initial identification of all C. auris isolates was performed using matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry [42 (link),43 (link)] by the APL—ProvLab. The molecular identity of these isolates was confirmed by amplifying and sequencing the Internal Transcribed Spacer (ITS) region of ribosomal DNA. The primers ITS-5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′) were used to amplify the ITS region (Integrated DNA Technologies). Genomic DNA was extracted using manual phenol–chloroform–isoamyl alcohol method [44 (link)]. The concentration of the extracted DNA was measured using a microvolume μDrop Plate (Thermo Fisher Scientific, Mississauga, ON, Canada, #N12391). The template and the primers were mixed in concentrations of 7.5 ng/μL and 0.25 μM, respectively, to a final volume of 10 μL. Sanger sequencing was then performed using a 3730 Genetic Analyzer (Thermo Fisher Scientific, Mississauga, ON, Canada, #A41046) at the Molecular Biology Services Unit at the University of Alberta. The resulting sequences were subjected to nucleotide BLAST analysis [45 ], which revealed 100% similarly to the standard strains. The C. auris isolates’ ITS sequences were submitted to NCBI with the accession number OP984814-OP984818.

The total RNA from the cells were extracted via a commercial extraction kit following the manufacturer’s instructions (Tri-Reagent™, Sigma-Aldrich, St. Louis, MO, USA). Depending on the quantified RNA content measured (μDrop Plate, Thermo Scientific, Waltham, MA, USA), first strand cDNA was synthesized and stored at −80 °C (prime Script™, Takara Bio Inc., Kusatsu, Shiga, Japan).