What is the optimal concentration for transfection?

There is no fixed optimal concentration for siRNA and ASO. Typically, siRNA is between 1-100 nM and ASO between 10-100 nM. Pre-experimental optimization is needed based on cell type, transfection method, and experimental objectives.

For cell lines that are easily transfected, lower concentrations can be selected; For cell lines that are difficult to transfect, it may be necessary to appropriately increase the concentration. It is recommended to set multiple concentration gradients and measure target mRNA silencing efficiency (qRT-PCR) 24-48 hours after transfection. Use the lowest possible concentration while ensuring effective silencing to reduce off-target effects and cytotoxicity.

How to handle a large number of cell deaths found during transfection?

• Adjust the concentration of transfection reagents. Transfection reagents themselves may have certain cytotoxicity, especially certain cationic liposomes or polymers.

• Change the culture medium promptly after transfection. Prolonged contact between the transfection complex and cells may cause continuous cell damage.

• Ensure cells are in a healthy growth state. The healthy state of cells is the foundation for resisting external stimuli. Before transfection, low-generation cells (< 20 generations), in the log-growth phase, with a fusion degree between 70-90% should be used. Cells in poor condition have significantly reduced tolerance to transfection agents.

• Optimize the ratio of transfection reagents to siRNA (N/P ratio). The balance between transfection efficiency and cytotoxicity largely depends on the charge ratio of the transfection reagent and the complex formed by the nucleic acid. An excessively high positive charge ratio leads to overly dense complexes, improving cell cell membrane efficiency while exacerbating cell membrane damage.

What could cause unobserved expected results?

• If the concentration is too low or the duration is insufficient, you can try increasing the concentration or extending the duration appropriately.

• Low transfection efficiency leads to insufficient intracellular siRNA/ASO content, so transfection conditions can be optimized, such as changing transfection reagents, adjusting reagent ratios, and cell density.

• If the target gene's mRNA level is too low, it is recommended that the Ct value of the target gene in the experimental cell line be below 30 before it can be used for knockdown experiments; otherwise, it is recommended to replace the experimental cell line.

• The target gene has a compensatory mechanism; even if its expression is suppressed, other genes may compensate for its function, resulting in a subtlety of the phenotype.

• siRNA/ASO degradation can be detected by methods such as agarose gel electrophoresis.

• siRNA/ASO sequence design is unreasonable and does not effectively bind target mRNA; the sequence design should be re-evaluated or replaced with a new sequence.

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