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.