2.4 RNAse digestion on plasma samples (RNAse protection assay)
To develop the digestion protocol, 100µl of Rhinovirus (HRV) RNA in Tris-Borate EDTA solution (8,5x108 copies/ml) and its scaling dilutions were treated with 2µl of RNase ONETMRibonuclease for two and a half hours as reported by Wu et al, 2012 [11]. HRV RNA quantification after RNAse digestion was performed with an in-house assay targeting 5’-UTR region.To investigate if the total amount of HCMV UL21.5 mRNA detected was packaged in virions or naked, RNAse digestion was performed on 600 µl plasma samples. After RNAse digestion, HCMV UL21.5 mRNA detection was performed on the ELITe InGenius® platform (ELITechGroup, USA) as previously described. To evaluate the actual effectiveness of RNAse digestion in plasma, the first 14 samples were added with 15µl of HRV RNA solution (6,7x106 copies/ml). After digestion, HRV RNA presence was investigated as described above.