Viral RNA was extracted from apical washes using the E.Z.N.A. viral RNA extraction kit (R6874-02; Omega Bio-tek, Inc., Norcross, GA, USA) and from tissue lysate using the E.Z.N.A. total RNA extraction kit (R6834-02; Omega Bio-tek, Inc., Norcross, GA, USA). For RNA quantification, primers and probes specific for each virus were used as previously described20 (link). The RNAseP housekeeping gene was used as an internal control for cell-associated virus quantification (4331182; Life Technology, Zug, Switzerland).
Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays were performed using the QuantiTect probe RT-qPCR kit (no. 204443; Qiagen, Valencia, CA, USA) in a StepOne Applied Biosystems thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). In each run and for each virus, four 10-fold serial dilutions of RNA reference standards were included. Results were analyzed using the StepOne version 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays were performed using the QuantiTect probe RT-qPCR kit (no. 204443; Qiagen, Valencia, CA, USA) in a StepOne Applied Biosystems thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). In each run and for each virus, four 10-fold serial dilutions of RNA reference standards were included. Results were analyzed using the StepOne version 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA).
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