ZIKV Envelope Gene Detection in Trophoblasts

The HC or trophoblasts were infected in the presence or absence of ruxolitinib as described previously and cells were harvested on day 6. Then RNA was reverse transcribed into cDNA and amplified in a one-step RT-PCR multiplex reaction with the LightCycler 480 RNA Master Hydrolysis Probe (Roche, Indianapolis, IN) using highly conserved sequences complementary to a 76 bp fragment from the ZIKV envelope gene as previously described [23 (link)] and an endogenous control (TaqMan Ribosomal RNA Control or beta globin Reagents; Applied Biosystems, Foster City, CA). The procedure was performed using the LightCycler 480 Instrument II (Roche). The analysis was performed by using the sample crossing point (Cp) value, which is the point at which an amplified product (the fluorescence of a sample rises above the background fluorescence) is visible. Relative quantification was determined using the ZIKV RNA Cp values relative to infected/untreated controls.