1) I knocked down a protein and collected cells 24 h and 72 h after transfection with Lipofectamine (to note, 5 h after transfection medium was replaced with medium with FBS).
2) KD efficiency was observed with qRT-PCR and it was successful for both time points (slighly better at 72 h).
3)Then I checked for expression of selected genes which I would express for them to be upregulated upon knockdown. And this was observed for the 24h time point. But 72 h after transfection (with a slight better KD efficiency!) there were a significant decrease in their expression when compared to the 24h.
Maybe another regulatory mechanism replaces my protein's role in supressing those genes. But does anyone know if longer exposure to siRNA within the cells might lead to off-target effects? I haven't found a correlation between siRNA exposure and off-target effects.
Thanks!
The gene silencing produced by siRNA is usually assessed 24 hours after transfection. The silence effect can last for 5-7 days. But the duration and level of gene knockdown depend on the cell type and the concentration of siRNA. To keep gene knockdown, transfection can be repeated.
learn more about, please visit https://transfection.bocsci.com/
- Badges
- Science method