Optimizing siRNA Transfection

Maximizing transfection efficiency while minimizing cytotoxicity are crucial for optimal gene silencing. Similar to balancing siRNA-induced knockdown and cell viability, there may also be a balance between siRNA delivery and downstream phenotypic assay conditions. It may be necessary to re-optimize siRNA delivery conditions for different downstream assays that are used in siRNA screening passes. The best transfection efficiencies are achieved for each cell type by identifying the following factors (in order of importance):

1. Choice of transfection reagent
2. Volume of transfection agent
3. Amount of siRNA
4. Cell density at the time of transfection
5. Length of exposure of cells to transfection agent/siRNA complexes
6. Transfection method: traditional transfection where cells are pre-plated or reverse transfection where cells are transfected as they adhere to the plate
7. Presence or absence of serum

Once the conditions for maximal gene silencing are determined, keep them constant among experiments with a given cell type.

1. Design and test two to four siRNA sequences per gene. Do not attempt to design siRNAs on your own. Use our custom siRNA builder to design your siRNAs.
2. Avoid RNases! Trace amounts of ribonucleases can sabotage siRNA experiments. Since RNases are present throughout the laboratory environment on your skin, in the air, on anything touched by bare hands or on anything left open to the air, it is important to take steps to prevent and eliminate RNase contamination. Thermo Fisher Scientific offers a complete line of products designed to detect and eliminate RNases.
3. Maintain healthy cell cultures and strict protocols for good transfection reproducibility. In general, healthy cells are transfected at higher efficiency than poorly maintained cells. Routinely subculturing cells at a low passage number ensures that there will be minimal instability in continuous cell lines from one experiment to the next. When performing optimization experiments, we recommend transfecting cells within 50 passages, since transfection efficiency drops over time.
4. Avoid antibiotic use. Avoid the use of antibiotics during plating and up to 72 hours after transfection. Antibiotics have been shown to accumulate to toxic levels in permeabilized cells. Additionally, some cells and transfection reagents require serum free conditions for optimal siRNA delivery. We suggest you perform a pilot transfection experiment in both normal growth media and serum-free media to determine the best condition for each transfection.
5. Transfect siRNAs using optimized reagents. Use an optimized siRNA transfection reagent and protocol for your cell type. The choice of transfection reagent is critical for success in siRNA experiments. It is essential to use transfection reagents formulated to deliver small RNAs (most commercially available transfection reagents were designed for large plasmid DNA, not small RNA molecules). Also, some reagents have been developed for the transfection of specific cell lines while others have broader specificity. For help selecting the appropriate transfection reagent, see siRNA transfection.
6. Use an appropriate positive control to optimize transfection and assay conditions. Housekeeping genes are suitable positive controls for most cell types. To optimize conditions, transfect target cells with several concentrations of an siRNA specific to your chosen positive control and to your experimental target siRNA. Measure the reduction in the control protein or mRNA level compared to untransfected cells 48 hours after transfection. Too much siRNA can lead to cell toxicity and death. For maximum convenience, Thermo Fisher Scientific offers positive control siRNAs against a variety of gene targets.
7. Use a negative control siRNA to distinguish non-specific effects. Negative controls should be designed by scrambling the nucleotide sequence of the most active siRNA. However, be sure to perform a homology search to ensure that your negative control sequence lacks homology to the genome of the organism being studied.
8. Use labeled siRNAs for protocol optimization. Fluorescently labeled siRNA can be used to analyze siRNA stability and transfection efficiency. Labeled siRNA is also useful to study siRNA subcellular localization and in double label experiments (with a labeled antibody) to visualize cells that receive siRNA during transfection and to correlate transfection with down-regulation of the target protein.

Visit Transfection Basics to learn more about performing transfection in your lab.