Ligthcycler 96

Viral RNA was isolated using RNeasy^® Plus Universal Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Quantification of viral RNA was performed using a real-time quantitative polymerase chain reaction (RT-qPCR) assay specific for the HTNV nucleocapsid coding region in a LigthCycler^® 96 (Roche, Mannheim, Germany) following the manufacturer’s instructions. Cells treated in parallel with the drugs were analyzed for viability using the CellTiter-Glo^® Assay System (Promega).

RNA preparation was performed using the Qiagen RNA extraction kit, according to manufacturer’s instructions. 1 μg of RNA was treated with RQ1 RNase-free DNase (Promega) for 2 hr at 37°C before heat-inactivation. cDNA synthesis was performed using the SuperScript III Reverse Transcriptase (Invitrogen), according to manufacturer’s instructions, in presence of oligo(dT)₂₀ (Invitrogen) and 500 ng of RNA. Real time PCR was performed using a LigthCycler 96 (Roche). Oligonucleotides MC057/058 and MC059/060 amplified ~120–150 bp fragments of TbDYRK or the YFP tag, respectively. Oligonucleotides MC055/056 (Ma et al., 2010 (link)), recognizing a fragment of GPI8, were used as an endogenous control for normalisation. PCRs were set up in triplicate, with each reaction containing 10 μL of Luna Universal qPCR Master Mix (New England BioLabs), 300 nM of each oligonucleotides, 5 μL of cDNA (diluted 1/10) in a final volume of 20 μL. PCR conditions were as follows: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 58°C for 1 min. Final melting curve was obtained by gradient increase temperature from 65°C to 95°C.
Ct values were normalised with the internal loading control GPI8. To allow relative quantifications, results were then compared to the mean value obtained for the DYRK gene in the WT strain, that has been set at 100% of expression.