Reliance one step multiplex rt qpcr supermix

Total RNA was isolated by using the Mag-Bind Viral RNA 96 kit (Omega Biotek; Norcross, GA, USA) on Kingfisher flex (Thermo Fisher Scientific; Waltham, MA, USA). Briefly, tick leg or mice brain or spleen tissues were placed in tubes containing 250 µl PBS-G (PBS with 0.5% gelatin, 30% rabbit serum and 1% 100× antibiotic–antimycotic [10,000 μg/ml of streptomycin and 25 μg/ml of amphotericin B]) and a stainless-steel ball bearing and macerated using a mixer mill. A 50-µl aliquot of homogenate was used for the RNA extraction following the protocol of the kit’s manufacturer (Omega Biotek). RT-qPCR was performed on a Bio-Rad C1000 Touch system with a CFX96 optical module using the Reliance One-Step Multiplex RT-qPCR Supermix (Bio-Rad Laboratories, Inc.; Hercules, CA, USA) with the following cycline parameters: reverse transcription at 55 °C, 15 min; then 95 °C, 10 min; followed by 95 °C/15 s and 60 °C/30 s for 40 cycles. The primer/probe set used to target the 3′-untranslated regions of POWV II was as previously reported [21 ]. Quantification of POWV II genome equivalents was performed using a previously developed assay with the same reagents and cycling parameters as described above [22 (link)].