Taqman fast virus mastermix

RNA was extracted with the QIAmp Viral RNA Kit (Qiagen) and RNA yields were measured with a Nanodrop 1000 (Thermo Scientific). Two separate assays were developed and validated, one for RESTV and one for EBOV, with primers and minor-groove binder probe sequences selected from previously described assays [33 (link)], to quantify the relative abundance of the EBOV and RESTV genome. The reaction was set up with 900 nM of forward and reverse primers, 250 nM probe, 1× TaqMan Fast Virus Mastermix (Life Technologies, London, United Kingdom), and tested using the ABi 7500 (Life Technologies) at a thermal sequence of 50 °C for 5 min, 95 °C for 20 s followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Samples were tested in triplicate and results analysed at a threshold value of 0.2. For quantification of viral genome copies, a synthetic RNA oligo was designed and developed (Integrated DNA Technologies, Germany). Concentrations were validated by qRT-PCR and Qubit Analyzer (Thermo Scientific) and a dilution range from 10⁹ to 10⁻¹ was used in duplicate.

RNA yields were measured with a Nanodrop 1000 (Thermo Scientific) and were normalised to 60ng/μl before addition to qRT-PCR. Primers and minor-groove binder probes were using previously described sequences16 (link) to quantify the relative abundance of the EBOV genome. The reaction was set up with 900 nM of forward and reverse primers, 250 nM probe, 1x TaqMan Fast Virus Mastermix (Life Technologies) to a total volume of 20 μl and tested using the ABi 7500 (Life Technologies) at a thermal sequence of 50 °C for 5 minutes, 95 °C for 20 seconds followed by 40 cycles of 95 °C for 3 seconds and 60 °C for 30 seconds Samples were tested in triplicate and results analysed at a threshold value of 0.2. For gene-specific RT-PCR profiling SYBR Green gene specific PCR assays were obtained as validated commercially available assays (Qiagen). RNA was treated with RNase free DNase (Promega) and cleaned up using the RNEasy MinElute Kit (Qiagen). RNA was equalised between all samples tested using nanodrop measurement (Thermo Scientific). Assays were performed in two stages, with the reverse transcription (RT) step performed with the RT2 First Strand kit (Qiagen) and the gene specific assay performed with gene specific primers (Qiagen) and the RT2 SYBR Green qPCR Mastermix (Qiagen), melt curves were performed to verify specificity of primer sets.