Transfection of antiviral siRNA/siLNA with Lipofectamine
3000 or peptide dendrimer KK-46
Vero E6 cells (ATCC®Number:
CRL-1586TM) were cultivated with 5 % of FBS (HyClone,
Logan, UT, USA) and 1 % PS in DMEM (PanEco, Moscow, Russia). The cells
were seeded into 24-well plates at a concentration of
105 cells/well in 0.5 mL of medium and incubated at
37°C, 5% CO2 for 12-24 h.
The medium was replaced with FBS-free Opti-MEM (Gibco, USA). Aliquots of
100 μL containing 0.5 μg of specific or non-specific siRNAs, 1.5 μL of
Lipofectamine3000 (Invitrogen) in Opti-MEM were added to VeroE6 cells.
After four hours, complexes were removed, and cells were infected with
SARS-CoV-2 at a multiplicity of infection (MOI) of 0.0001 and incubated
for 1 hour at 37°C, 5% CO2. Cells were washed and
non-adherent virus was removed and re-suspended in the fresh media. Cell
culture supernatants and RNA lysates were harvested at times indicated
and stored at -80 °C.
The same procedure was used for transfection with siRNA/KK-46 complexes.
The only exception was the amount of the components. Three increasing
concentrations of complexes 21, 42 and 84 μg/per well which included the
sums of KK-46+siRNA/siLNA, μg: 20+1, 40+2 and 80+4, respectively (20:1
weight ratio) were used. Unspecific siRNA targeting the gene of firefly
luciferase (siLuc) and SARS-CoV-2 infected cells were used as negative
controls.