I transfected my siRNA and I'm seeing less than 75% silencing at the mRNA level. What is wrong?
The two most frequent causes of poor silencing are low transfection efficiency and poor siRNA sequence design. Often the lack of silencing is related to inefficient delivery of siRNA into the cells, suggesting that transfection conditions need to be optimized. If these procedures to optimize transfection conditions have already been performed and poor or moderate silencing persists, an alternative transfection reagent or technique (e.g., electroporation) may provide better delivery for that particular cell line.
If transfection efficiencies have been optimized and poor silencing results are still observed, the siRNA sequence design may be the cause. Conventional siRNA design rules and publicly available design tools vary significantly in their ability to select functional sequences for difficult-to-silence genes. In addition, poor to moderate siRNA duplexes have been shown to exhibit variability in silencing efficiency. If poor silencing due to siRNA sequence design is suspected, siRNAs designed with Dharmacon's SMARTselection and SMARTpool technologies are highly recommended for optimal efficiency.