A New Way to Assess siRNA Delivery Efficiency
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Efficient and reproducible transfection is critical for any siRNA experiment. Transfection conditions should be optimized to provide the highest levels of gene silencing while minimizing toxicity associated with the transfection process. Ambion’s Silencer GAPDH siRNA is an ideal positive control for transfection optimization studies because it efficiently silences GAPDH--a ubiquitously and highly expressed gene--in human, mouse, and rat cells. Now Ambion has developed a new assay to rapidly measure this control’s efficiency of delivery and induced level of GAPDH knockdown: the KDalert GAPDH Assay Kit.
Many researchers are currently using quantitative RT-PCR or Western blotting to assess siRNA-induced knockdown. While accurate, these techniques can be time-consuming and labor intensive. Another means to monitor siRNA delivery is use of fluorescently labeled siRNA. This method has the advantage of speed, but it can be unreliable since siRNAs can be trapped in endosomes or other sub-cellular compartments that keep them from directing target mRNA cleavage.
Many researchers are currently using quantitative RT-PCR or Western blotting to assess siRNA-induced knockdown. While accurate, these techniques can be time-consuming and labor intensive. Another means to monitor siRNA delivery is use of fluorescently labeled siRNA. This method has the advantage of speed, but it can be unreliable since siRNAs can be trapped in endosomes or other sub-cellular compartments that keep them from directing target mRNA cleavage.
One Simple Assay for Two Critical Readouts
The new KDalert GAPDH Assay Kit relies on a simple fluorescence-based assay to quickly and accurately measure GAPDH enzyme activity in cell lysates. By comparing GAPDH enzymatic activity in cultures transfected with GAPDH siRNA to those transfected with a negative control siRNA, the level of gene silencing, and thus the success of the transfection, can be readily determined. In addition, transfection conditions can be evaluated for cellular toxicity using the KDalert GAPDH Assay Kit. For this type of analysis, cells are transfected with a negative control siRNA
(e.g., Silencer Negative Control #1) followed by evaluation of GAPDH activity relative to untransfected cell; reduced GAPDH activity is an indication that transfection induced cytotoxicity. Because the assay can be used to assess both GAPDH siRNA-induced knockdown and transfection-induced toxicity, researchers can use the assay in conjunction with Silencer GAPDH and Negative siRNA controls to rapidly identify optimal transfection conditions that result in efficient delivery without compromising cell viability (Figure 1).
Figure 1. Comparison of GAPDH Enzyme Activity and qRT-PCR Data. Varying cell numbers of three cell lines (A549, HeLa, SK-N-AS) were transfected with Silencer® GAPDH siRNA and Silencer Negative Control #1 siRNA. GAPDH enzyme activity, using the KDalert™ GAPDH Assay, and GAPDH mRNA, by qRT-PCR, were measured 48 hr after transfection. For each condition, the remaining expression for GAPDH siRNA transfected cultures was calculated as a percentage of expression of negative control siRNA-transfected cultures.
(e.g., Silencer Negative Control #1) followed by evaluation of GAPDH activity relative to untransfected cell; reduced GAPDH activity is an indication that transfection induced cytotoxicity. Because the assay can be used to assess both GAPDH siRNA-induced knockdown and transfection-induced toxicity, researchers can use the assay in conjunction with Silencer GAPDH and Negative siRNA controls to rapidly identify optimal transfection conditions that result in efficient delivery without compromising cell viability (Figure 1).
Figure 1. Comparison of GAPDH Enzyme Activity and qRT-PCR Data. Varying cell numbers of three cell lines (A549, HeLa, SK-N-AS) were transfected with Silencer® GAPDH siRNA and Silencer Negative Control #1 siRNA. GAPDH enzyme activity, using the KDalert™ GAPDH Assay, and GAPDH mRNA, by qRT-PCR, were measured 48 hr after transfection. For each condition, the remaining expression for GAPDH siRNA transfected cultures was calculated as a percentage of expression of negative control siRNA-transfected cultures.
One Simple Assay for Two Critical Readouts
The KDalert GAPDH Assay Kit procedure is both simple and fast. To use, add the included cell lysis buffer to cells two to three days post-transfection, incubate for 20 minutes, add the diluted master mix of assay reagents, and then read the increase in fluorescence four minutes later in a microplate or standard fluorometer (Figure 2). The entire procedure can be completed in about 30 minutes with minimal sample handling steps.
Figure 2. KDalert™ GAPDH Assay Kit Protocol Schematic.
Figure 2. KDalert™ GAPDH Assay Kit Protocol Schematic.
Compatible Across Cell Types and Over a Wide Dynamic Range
The KDalert GAPDH Assay Kit includes enough reagents for 375 assays (300 assays + 3 standard curves). The 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 plated in 96 well plates 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, including: Silencer GAPDH and Negative Control siRNAs, Silencer CellReady Transfection Optimization Kit, siPORT NeoFX Transfection Agent, and the Silencer siRNA Transfection II Kit.
Scientific Contributors
Luis Foncerrada, Mike Byrom, Leslie Beauchamp, Lance Ford,
Joe Krebs • Ambion, Inc.
Scientific Contributors
Luis Foncerrada, Mike Byrom, Leslie Beauchamp, Lance Ford,
Joe Krebs • Ambion, Inc.
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