Because siRNA differs in size and structure from plasmid DNA, transfection reagents can be optimized and formulated differently for delivery of these RNA molecules. After selection of the appropriate transfection reagent, optimization of several experimental parameters is key to achieving the highest efficiency siRNA transfection and corresponding effective knockdown of target gene expression.
Below, we highlight our top “Tips from the Bench” for optimizing siRNA delivery. Note: most of these tips are applicable to delivery of other classes of oligos, including but not limited to miRNA and antisense oligonucleotides.
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 reagent amount 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.
Transfect a non-targeting or negative 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.
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® 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.
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.
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.