Results related to Figure 1A - Healthy Blood Donors (HBD)
Description
The level of HERV-W ENV and HERV-K ENV mRNA in PBMC cultures from HBD, exposed to SARS-CoV-2 (MOI:0.1) or mock treated (culture medium), was analyzed by RT-qPCR. The graph presents the mean results from triplicate (7 out 11 donors) or single cultures (4 out 11 donors) at 2h post-exposure.
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RT-qPCR was performed using specific primers for HERV-W and HERV-K envelope genes, as already validated in patients with HERV-associated diseases(Faucard et al., 2016; Levet et al., 2017; Li et al., 2015), using B2M mRNA as a suitable reporter gene for PBMC (Milhem et al., 2020). For in vitro analyses, cells were harvested at several time points after exposure to SARS-CoV-2 virus or protein, and total RNA extracted. For blood samples, freshly isolated PBMC were collected to similarly extract RNA. 200 ng of DNase-treated RNA were reverse-transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad, 1708891) according to the manufacturer’s protocol. A control with no-RT was prepared in parallel, to confirm the absence of contaminating DNA in PCR experiments. An amount of 5 ng of initial RNA in RT reaction has been used to quantitatively evaluate the transcriptional levels of HERV-W ENV, HERV-K ENV, N SARS-CoV-2 (Ferren et al., 2021) and ACE2 genes by RT-qPCR). The assays were performed in a StepOnePlus instrument (Applied Biosystems) using Platinum SYBR Green (Invitrogen, 11744-500). B2M was used as housekeeping gene.