Cellular proliferation

In order to determine cellular proliferation, a BrdU incorporation assay was used. The Huh-7 cells were seeded 24 h prior to transfection into black 96-well plates and transfected with 12.5 ng oligonucleotides (according to the HiPerfect protocol; Qiagen GmbH) with an initial constant cell count of 5×10⁴ cells/well. At 48 h post-oligonucleotide transfection, the cells were labeled with BrdU labeling reagent for 4 h (with a final concentration of 100 µM) using the Cell Proliferation ELISA kit (Roche Applied Science, Penzberg, Germany). The cells were then fixed using FixDenate for 30 min and incubated with Anti-BrdU POD (with a final concentration of 10 µM) for 90 min, as previously described (20,25).

All cellular proliferation experiments were performed in quadrate and repeated 5 times.