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KDalert™ GAPDH
Assay Kit
A New Way to Assess
siRNA Delivery Efficiency
• Assess GAPDH siRNA delivery
in less time for a fraction of 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 Transfection Optimization Kit
for a complete transfection optimization solution
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.
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).
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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.
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Rapid, Time-saving Procedure
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.
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| 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. For more information
about how these products synergize, see Complete Toolkit for Transfection
Optimization.
Scientific Contributors
Luis Foncerrada, Mike Byrom, Leslie Beauchamp, Lance Ford,
Joe Krebs • Ambion, Inc.
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