Cellular Behavior and Response Varies with Passage Number
Maintain a similar passage number between experiments to ensure reproducibility. A low passage number can make cells more sensitive to transfection whereas a high passage number can render cells refractory to transfection.
Use healthy, actively dividing cells to maximize transfection efficiency. Mirus Bio recommends plating cells the night before a transfection experiment at a density that will promote cell division to obtain 50-70% confluency for transfection the following day. Lower cell densities may be necessary when post-transfection incubation times are greater than 48 hours. If lower cell densities are plated, test a range of TransIT-TKO® reagent to determine optimal concentration.
Dilute siRNA using the manufacturer’s recommended buffer. Alternatively, use 100 mM NaCl in 50 mM Tris, pH 7.5, made with RNase-free water. Do not use water alone to dilute siRNA, as this may result in denaturation of the siRNA.
siRNA used for transfection should be highly pure, sterile, and the correct sequence. Depending on the type of experiment, the optimal final siRNA concentration for transfection is typically within the range of 10-50 nM. As a starting point, we recommend 25 nM siRNA (final concentration in well).
Transfect a non-targeting or non-sense siRNA control sequence to verify that the gene expression knockdown or phenotype is attributed to the gene-specific siRNA. Additionally, targeting a gene with multiple siRNA sequences ensures that the resulting phenotype is not due to off-target effects.
Use an Optimal Volume of Transfection Reagent
When working with a new cell type, test a range of reagent volumes at a fixed siRNA concentration to find the level that achieves the highest transfection efficiency with minimal toxicity. For optimization, test three levels of TransIT-TKO® Reagent, e.g. 1, 2.5, and 4 μl per well of a 24-well plate, using 25 nM siRNA (final concentration in the well). It may be necessary to titrate outside of this range depending on the cell type.
Complex Formation Time
After mixing the siRNA and transfection reagent, incubate to form complexes for 15-30 minutes at room temperature, before adding the mix to your cells. Transfection efficiency may decrease if the complex formation exceeds an hour.
Post-transfection Incubation Time
Determine the optimal incubation time by testing a range from 24-72 hours post-transfection, depending on the stability of the target mRNA and its encoded protein. When quantifying knockdown efficiencies at the mRNA level, assaying at 24 hours post-transfection is often sufficient. When quantifying knockdown efficiencies at the protein level, longer post-transfection incubation may be necessary, particularly if the target protein has a long cellular half-life.