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Tools for Optimizing siRNA Delivery: Define the Best Conditions for siRNA Delivery in Cultured Cells
Optimization of siRNA delivery to cultured cells, whether by transfection or electroporation, is critical for obtaining reliable and reproducible data in gene silencing experiments. Ambion siRNA delivery optimization kits and reagents (Figure 1) are easy to use and include detailed instructions to help you streamline the optimization process.
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| Figure 1. Tools for Optimizing siRNA Delivery. |
siRNA Transfection
The Silencer® siRNA Starter Kit is the recommended kit for those just starting siRNA experiments, as it features a convenient package of control siRNAs, siRNA transfection agent, and downstream detection reagents. The included siPORT™ NeoFX™ Transfection Agent effectively delivers siRNAs to a broad range of cell lines, and supports both reverse and forward (“pre-plating”) transfection protocols. The kit also includes a well-characterized Silencer GAPDH siRNA, which has been shown to strongly down regulate the GAPDH gene in human, mouse, and rat cells (see sidebar, Why Use GAPDH Control siRNAs?), as well as a non-targeting negative control siRNA known as Silencer Negative Control #1 siRNA. Finally, the kit includes three different systems for monitoring GAPDH siRNA induced knockdown. An Anti-GAPDH antibody is provided to assess GAPDH protein levels by Western blot or immunofluorescence. The Ambion KDalert™ GAPDH Assay is also provided to quickly measure GAPDH enzymatic activity. This assay has the added benefit that it can be used to assess cell viability. Finally, RT-PCR primers, for real-time quantitative RT-PCR detection of GAPDH mRNA are included.
The Silencer siRNA Transfection II Kit is the ideal choice for optimizing siRNA transfection conditions in any size plate. This kit includes both siPORT Amine and siPORT NeoFX Transfection Agents for broad cell line compatibility. Both transfection agents support traditional “pre-plating” transfection protocols as well as reverse transfection procedures, where cells are transfected during subculturing. Transfection efficiency is monitored using Silencer GAPDH siRNA and the non targeting Silencer Negative Control #1 siRNA. Because two transfection agents are included, the Ambion Silencer siRNA Transfection II Kit makes it easy to identify the appropriate siRNA delivery agent and conditions for your cell type.
The Silencer CellReady™ siRNA Transfection Optimization Kit was developed specifically to accelerate siRNA transfection optimization in 96-well plates. This kit comes with three 96-well plates containing pre-plated positive control siRNA targeting human, mouse, and rat GAPDH and nontargeting Negative Control siRNA. It also includes siPORT NeoFX Transfection Agent. The convenient arrangement of siRNAs on each plate can be used to assess the effectiveness of up to 24 transfection conditions in duplicate.
Detailed instructions included with all three siRNA transfection kits help new users test important variables, such as choice of transfection agent, transfection agent volume, cell number, timing of a potential post-transfection wash, and timing of the post-transfection assay.
siRNA Electroporation
For difficult-to-transfect cell types (e.g., suspension cells or primary cells), the siPORT™ siRNA Electroporation Kit provides an efficient way to test and optimize electroporation parameters. This kit contains siPORT siRNA Electroporation Buffer, which is designed to emulate the natural cytoplasmic composition of cells, to facilitate rapid pore resealing, and to promote high cell viability after electroporation. Ambion Scientists in our R&D laboratories have successfully used this unique buffer with the Bio-Rad® Gene Pulser Xcell™, the BTX® ECM830, and the PA-4000S PulseAgile™ pulse generators. In addition to GAPDH Control siRNA and Negative Control siRNA, Cy™3 dye-labeled Control siRNA is also included that enables monitoring of siRNA uptake into target cells by fluorescence microscopy. A detailed instruction manual helps new users optimize electric field strength, pulse strength, and number of field pulses to ensure maximum delivery of siRNA by electroporation in standard 1 mm cuvettes.
Quickly Assess siRNA Delivery and Cell Viability in the Same Assay
The efficiency of siRNA delivery is most frequently monitored by measuring the reduction in GAPDH mRNA and/or protein levels in cells transfected with GAPDH siRNA relative to cells transfected with negative control siRNA.
The KDalert™ GAPDH Assay Kit simplifies this step by providing researchers with a reliable measure of GAPDH enzyme activity in cultured human, mouse, or rat cells. With this straightforward assay, which can be completed in under 30 minutes, researchers use a microplate fluorometer for quantitation (an absorbance reader can also be used). In addition to its use for rapid monitoring of GAPDH siRNA induced knockdown, the assay can also be used in the same experiment to monitor transfection agent induced toxicity (Figure 2). For this analysis, GAPDH activity from negative control siRNA transfected cells is compared to that of untreated cells. The KDalert GAPDH Assay Kit integrates seamlessly with all of the Ambion transfection optimization products listed in this article.
For more information on optimizing siRNA delivery conditions, visit the siRNA Delivery Resource.
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| Figure 2. Optimizing Transfection Conditions Using the KDalert™ GAPDH Assay Kit. HeLa cells were transfected with either GAPDH or Negative Control #1 siRNA using the indicated amount of siPORT™ NeoFX™ Transfection Agent. 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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