- Assess GAPDH siRNA delivery in 1/3 the time for 1/3 the cost of real-time PCR
- Analyze from one to 96 samples simultaneously
- Measure both GAPDH siRNA induced knockdown AND transfection induced toxicity
- Use with Silencer® GAPDH siRNA Controls and Silencer® CellReady™ siRNA Transfection Optimization Kit for a complete transfection optimization solution
The first step of any siRNA experiment is to work out appropriate siRNA delivery conditions for the particular cell type being used. Traditionally, siRNA delivery efficiency is monitored by transfecting cells with a positive control siRNA and then measuring the subsequent reduction of target mRNA levels by real-time PCR or the reduction of target protein levels by Western blotting. While accurate, these techniques are time consuming and labor intensive. In addition, neither of these techniques effectively monitors transfection reagent induced cytotoxicity.
With the KDalert GAPDH Assay Kit, you can obtain a reliable measure of GAPDH enzyme activity in cultured human, mouse, or rat cells in less than 30 minutes using a microplate fluorometer. The KDalert GAPDH Assay Kit, which also measures transfection induced cytoxicity, is designed for use with Ambion’s Silencer® GAPDH siRNA. GAPDH is a ubiquitously and highly expressed gene, making this siRNA an ideal positive control for transfection optimization experiments in human, mouse, and rat cells.
Rapid, Time-saving Procedure
The KDalert GAPDH Assay Kit relies on a simple fluorescence-based assay to quickly and accurately measure GAPDH enzyme activity in cell lysates (Figure 2). Use the assay to optimize siRNA transfection by transfecting individual cell samples with a GAPDH siRNA and a negative control siRNA. Two to three days after transfection, simply add the included cell lysis buffer to the cells, incubate for 20 minutes, add the diluted master mix of assay reagents, and then read the increase in fluorescence four minutes later using a microplate or standard fluorometer. The assay procedure can be completed in about 30 minutes with minimal sample handling. Additional cell lysis buffer can be purchased separately.
One Simple Assay for Two Critical Readouts
Once the assay is complete, siRNA delivery efficiency is assessed by comparing GAPDH activity levels in the GAPDH siRNA and negative control siRNA treated samples (Figure 3). Because GAPDH is expressed at relatively constant levels, the assay can be used in the same experiment to monitor transfection agent induced toxicity. For this analysis, GAPDH enzyme activity from negative control siRNA transfected cells is compared to that of untreated cells. Reduced GAPDH activity in negative control transfected cells compared to non-transfected cells is an indication that the transfection induced cytotoxicity.
Compatible Across Cell Types and Over a Wide Dynamic Range
The KDalert GAPDH Assay Kit works in a wide variety of cell types over a broad range of cell culture conditions. The fluorometric nature of the assay endows it with high sensitivity and a wide dynamic range, permitting accurate determination of GAPDH activity in samples consisting of 2,000–12,000 cells/well using the standard protocol, and with as few as 500 cells/well or >12,000 cells/well using a slightly modified assay procedure.
Part of Ambion's Complete Solution for siRNA Transfection and Transfection Optimization
The KDalert GAPDH Assay Kit integrates seamlessly with other Ambion products used for siRNA transfection and transfection optimization, such as: Silencer® GAPDH Control siRNAs, Silencer® Negative Control siRNAs, Silencer® CellReady Transfection Optimization Kit, siPORT™ NeoFX™ Transfection Agent, and the Silencer® siRNA Transfection II Kit.
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