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 are easy to use and include detailed instructions to help you streamline the optimization process.
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.
Quickly Assess siRNA Delivery and Cell Viability in the Same Assay
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.
- Optimize transfection efficiency by transfecting a GAPDH positive control siRNA using experimental transfection agents and conditions to identify which provide the maximum knockdown of GAPDH.
- Optimize cell viability by transfecting a negative control siRNA to identify the transfection agent and conditions that do not significantly decrease cell number compared to untransfected cells.