RT-qPCR Assay
Viral RNA was extracted from plasma and sperm of recovered patients using RNA with Quick-RNA MiniPrep (ZYMO RESEARCH, Irvine CA, catalog #1054) according to the manufacturer’s guidelines. Genomic DNA contamination was removed with DNase-Free DNase set (ZYMO RESEARCH, catalog #E1010). The total RNA extracted was stored at -80°. Then, 500ng RNA was retro-transcribed with ImProm-II Reverse Transcriptase kit (Promega) following manufacturer’s instruction and random primers (0,5 ug/ul). qPCR assay was performed with Luna Universal qPCR Master Mix (New England Biolabs) on 10 ng cDNA template/reaction using exon-spanning primers (final concentration 1 μM). ΔΔCT and 2^-ΔCT methods were used to calculate the expression levels, samples were normalized to GAPDH.