Quantinova reverse transcription kit

Total RNA samples from P. patens were isolated with RNeasy Plant Mini Kit according to manufacturer’s instructions (Qiagen, Germany). For cDNA synthesis, 2 μg of total RNA were reverse transcribed with QuantiNova Reverse Transcription (RT) Kit (Qiagen, Germany). To estimate amounts of cDNA templates of the selected genes, quantitative RT-PCR assays were performed using specific primers listed in Supplementary Table 2, designed by Primer3Plus (Untergasser et al., 2012 (link)). qPCR was performed in an Applied Biosystems StepOne real-time PCR system. Each 10 μL reactions contained 5 μL of SYBR Green PCR Master mix (2 X), 0.5 μM primers mix and 2 μL of template cDNA (1/10 dilution). The thermocycler was programmed to run for 5 min at 95°C, followed by 40 cycles of 15 s at 94°C, 30 s at 60°C. Transcript accumulation of each gene was normalized relative to the constitutively expressed E3 ubiquitin ligase (Le Bail et al., 2013 (link)). Amplification efficiencies of the different primer combinations were all > 90%. Relative expression was determined using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Each data point is the mean value of three biological replicates. Two technical replicates were used for each sample.

Forty fly heads per each genotype were collected on dry ice and total RNA was extracted using TRIZOL (ThermoFisher Scientific) according to the manufacturer’s protocol. RNA concentration and quality were analyzed spectrometrically. Reverse transcription of RNA was performed with 2–4 µg RNA using QuantiNova^TM Reverse Transcription kit (Qiagen) according to the manual. Sample dilution for quantitative PCR was determined by standard curves using dilution series of sample mixes using the Applied Biosystems StepOnePlus^® (Thermo Fisher) with Biozym Blue S'Green qPCR Kit Separate ROX (Biozym) or QuantiNova PCR Kits (Qiagen). PCR efficiencies were within 85–130% for all PCR reactions. The efficiencies of the primers were included in the calculation (Nolan et al, 2006 (link)). Target mRNA quantification was calculated by relative ΔCt comparison (ΔΔCt‐method) using Rpl32 and αTubulin mRNA as internal standards (revealing similar results) and subsequently normalized to signals from wild‐type fly heads. Primers used for the RT–PCR are listed in Appendix Table S2.

To detect fecal viral shedding after the viral challenge, feces collected from rectal swabs were resuspended in 900 µL of DPBS (Gibco) and mixed using a vortex. The resuspended samples were centrifuged at 13,793× g for 10 min. The viral RNA was extracted using the Cador^® Pathogen 96 QIAcube^® HT Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Reverse transcription was performed using the QuantiNova^TM Reverse Transcription Kit (Qiagen) to synthesize cDNA for subsequent quantitative real-time PCR, as described previously [49 (link)]. The detection limit of the assay was 4.7 log₁₀ RNA copies per mL based on the standard curve of the in vitro transcribed PEDV RNA.

MW001 and MW413 were grown in triplicate in medium supplemented with 0.1% N-Z-Amine or 0.1% N-Z-Amine and 0.2% sugar (L-arabinose, D-arabinose, D-xylose, dextrin, sucrose or glucose). Cell cultures (14 ml) were harvested at mid-exponential phase (OD₆₀₀ ∼ 0.4). RNA was isolated as described previously (Lassak et al., 2012 (link)). cDNA synthesis was carried out with QuantiNova^TM Reverse Transcription Kit (Qiagen, Venlo, Netherlands) according to the manufactures protocol. The quantitative RT-PCR was performed in the Magnetic Inducible Cycler (MIC) (Bio molecular systems, Upper Coomera, Australia) with 2xqPCRBIO SyGreen mix Lo-ROX (PCRBiosystems, London, United Kingdom) by following the manufacturer’s instruction. Probes were activated by heating to 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. The transcription levels were analyzed with gene-specific primers (Supplementary Table S3) for the sugar binding protein (saci_2122). The cq-values were normalized to the cq-values of the housekeeping gene secY (saci_0574).

Fresh tissues were removed and immersed in RNAlater^TM Solution (Thermo Fisher Scientific, Cat# AM7020). Total RNA was extracted with TRIzol^TM reagent (Thermo Fisher Scientific, Cat# 15596018). cDNA was prepared from total RNA using a QuantiNova^TM reverse transcription kit (Qiagen, Dusseldorf, Germany, Cat# 205411) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using a QuantiNova^TM SYBR^® Green PCR kit (Qiagen, Cat# 208054) on an Applied Biosystems^® 7500 Real-Time PCR System according to the manufacturer’s instructions. The primers are shown in Supplementary Table S2.

500 ng of total RNA was converted into cDNA using QuantiNovaTM Reverse Transcription Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. The utilized kit includes a genomic DNA elimination step. Reverse transcription quantitative PCR (RT-qPCR) was performed using the LightCycler®480 (Roche, Mannheim, Germany). A 20 μL reaction contained 10 µl 2 × QuantiTect SYBR Green PCR Master Mix (QIAGEN, Hilden, Germany), 3 μL ddH₂O, 6 μL cDNA template, and 2.5 μM of each primer. The primer sequences are provided in Supplementary Table S2. The following thermal conditions for amplification were applied: 95 °C for 15 min, followed by 45 cycles at 94 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s, and final 72 °C for 5 min. Melting curves were obtained by slow heating (0.5 °C/s) at temperatures in the range of 65 to 95 °C. The 2^-ΔΔCT method was performed for relative quantification analysis using the housekeeping genes PEX16 and FPGS^{44 (link)}. For PEX16, the PCR product was analyzed using Fragment Analyzer (Agilent, Santa Clara, California, USA) to ensure that only a single PCR product was obtained (Supplementary Figure S6). Samples were excluded from RT-qPCR if less than 400 ng of RNA remained after NGS. This resulted in a reduced sample size of n = 33 (survivors n = 18 (day 0 n = 8, day 7 n = 10), non-survivors n = 15 (day 0 n = 9, day 7 n = 6).

qRT-PCR was performed using RNA isolated from C. neoformans at day three of melanization. cDNA was prepared using the QuantiNova^TM Reverse Transcription kit (Qiagen, Hilden, Germany) using 1 μg of isolated RNA. cDNA, primers, and SYBR green were added to a 96-well PCR plate that amplified the products using a CFX96 Real-time PCR Detection System (BioRad Laboratories, Hercules, CA, United States). Each cDNA treatment was performed in quadruplicate and normalized using the geometric mean of the housekeeping genes UBC6 and TFC1. Fold changes were determined using the 2^–ΔΔC′T method, as described by Livak and Schmittgen (2001) (link).