Maximize Gene Knockdown, Minimize Toxicity
Efficient, reproducible siRNA delivery is essential for successful RNAi experiments. Because siRNA delivery is so critical, it is highly worthwhile to invest the time and effort to determine the best siRNA delivery method and conditions for each cell line studied. Careful optimization of procedures limits siRNA delivery variability and enhances the quality of the results.

The best siRNA delivery protocol provides good gene knockdown (i.e., effective siRNA delivery), while maintaining an acceptable level of cell viability (i.e., low cytotoxicity).

Some of the conditions that improve gene knockdown, such as higher amounts of transfection agent, also result in decreased cell viability. Therefore, it is important to assay for both gene knockdown and cell viability when developing siRNA delivery conditions.  

Controls Are Key
Negative controls that do not target any endogenous transcript are needed to control for non-specific effects on gene expression caused by introducing any siRNA. Easy-to-assay positive controls are needed to optimize transfection conditions, ensure that siRNAs are delivered, and ascertain that a particular downstream assay is working. Because positive controls are required for many different aspects of an RNAi experiment, often more than one control is required.

Ambion scientists use and recommend a number of different controls for siRNA experiments. Most of these coincide with the suggested controls detailed in an editorial published in Nature Cell Biology [Whither RNAi? (2003) Nature Cell Biology 5:489–90].

Transfection Agent
In this experiment, we chose siPORT™ NeoFX™ Transfection Agent as our siRNA delivery vehicle. This transfection agent efficiently delivers siRNA into a variety of cell types with minimal cytotoxicity compared to other commercially available transfection reagents. It is serum tolerant, compatible with reverse transfection, and yields highly reproducible results.

siRNA Controls
For optimizing transfection conditions, we typically use Silencer® GAPDH siRNA and Silencer Negative Control #1 siRNA. Both of these siRNAs have been extensively validated for use in human, mouse, and rat cells. GAPDH mRNA is a particularly useful target for a positive control siRNA for use in transfection optimization experiments because it is ubiquitously expressed across cell types and is easy to assay. Silencer Negative Control #1 siRNA has been tested in multiple cell lines and found to have minimal effects on cell survival or cell proliferation. This control is useful in essentially all siRNA experiments to control for the effects of delivering any siRNA.

Optimization Process and Results
To determine the best transfection conditions to use in our HeLa cell system, we transfected Silencer GAPDH siRNA or Silencer Negative Control #1 siRNA using increasing amounts of siPORT NeoFX  Transfection Agent (Figure 2). We then analyzed GAPDH mRNA levels by qRT-PCR to monitor siRNA delivery efficiency, and examined relative cell numbers using the KDalert™ GAPDH Assay Kit to monitor transfection induced cytotoxicity. Based on these data, as well as on additional experiments not shown, we decided to use 0.3 µL siPORT NeoFX reagent for subsequent experiments.

Figure 2. Optimizing Transfection Conditions with siPORT™ NeoFX™ Transfection Agent. HeLa cells were transfected with either GAPDH or Negative Control #1 siRNA using the indicated amount of siPORT™ NeoFX™ Transfection Agent (Cat. #AM4510). GAPDH mRNA levels were measured 48 hrs after transfection using a corresponding TaqMan Gene Expression Assay. Percent remaining gene expression is depicted versus negative control siRNA-treated cells. Percent cell viability was measured with the KDalert™ GAPDH Assay Kit and is shown relative to non-transfected cells.

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