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KDalert™ GAPDH
Assay Kit
Quickly Assess siRNA Delivery and Cell Viability
in the Same Assay
Transfection optimization is critical to performing
meaningful siRNA experiments in cultured cells. The GAPDH (glyceraldehyde
3-phosphate dehydrogenase) gene is an abundant and ubiquitously
expressed “housekeeping” gene that can be manipulated
and measured providing two-fold utility for siRNA optimization
experiments in cultured cells. First GAPDH gene expression can
be readily knocked down in many cell types by delivery of the
validated Silencer® GAPDH siRNA. The efficiency
of siRNA delivery can be easily monitored by measuring the reduction
in GAPDH protein levels in cells transfected with GAPDH siRNA
relative to cells transfected with negative control siRNA. Second,
GAPDH expression can serve as a marker for identifying cellular
toxicity resulting from transfection.
A Rapid, Convenient Assay
Ambion has developed a rapid, sensitive assay
system to quantitate siRNA induced GAPDH knockdown (KD) in cultured
cells. This assay, provided in the KDalert™ GAPDH Assay
Kit, is extremely simple and easy to perform (see sidebar, KDalert:
A Simple 3-Step Procedure). It allows the direct measurement
of GAPDH enzymatic activity in cell lysates using a fluorometer
or spectrophotometer. The entire procedure can be performed in
about 30 minutes and is compatible with all types of cultured
animal cells. Monitoring changes in GAPDH levels can serve as
an excellent assessment of GAPDH siRNA transfection efficiency.
GAPDH levels can also be quantitated to monitor transfection
induced changes in cell viability by comparing GAPDH levels in
nontransfected versus negative control siRNA transfected samples.
The KDalert assay, thus, provides a convenient and cost-effective
method to quantitate the efficiency of siRNA delivery into cells
and to simultaneously monitor cell viability.
Easy Transfection Optimization
The KDalert assay is an integral part of Ambion’s
suite of products to optimize siRNA transfection into cells.
Of particular note are the Silencer® CellReady™ siRNA
Transfection Optimization Kit and the Silencer® siRNA
Transfection II Kit, which can be used to deliver GAPDH
and negative control
siRNAs into cells under a range of transfection conditions. The KDalert Kit
can then rapidly identify transfection conditions that maximize silencing while
minimizing transfection-associated cytotoxicity. When used together, these
kits enable researchers to rapidly determine optimal transfection conditions,
which can then be used for all subsequent transfections into that cell type.
(The Silencer® siRNA Starter Kit, which combines these two products
for researchers new to RNAi, is also available).
Rapid Measurment of Silencing and
Cell Viability
To demonstrate the ease of siRNA delivery optimization
afforded by combining the Silencer CellReady siRNA Transfection
Optimization Kit and the KDalert GAPDH Assay Kit, we used the
two kits to optimize siRNA delivery into HepG2 cells, a cell
type that can be difficult to transfect. We created a matrix
of transfection conditions in a 96 well Silencer CellReady
siRNA Transfection Optimization Plate in which cell number and
the amount of siPORT™ NeoFX™ Transfection
Agent (included in the kit) were varied. HepG2 cells were reverse
transfected with either Silencer GAPDH siRNA or Silencer Negative
Control #1 siRNA; these siRNAs are pre-aliquotted into plates
as part of the Silencer CellReady siRNA Transfection
Optimization Kit. Two days after transfection, the medium was
removed and the cells were lysed using the reagents and protocol
in the KDalert Kit. GAPDH activity in each of the lysates was
then determined using the KDalert assay.
Figure 1 indicates that while GAPDH knockdown
was observed for all transfection conditions tested, the best
knockdown was observed using 0.6 µl siPORT NeoFX per
well. These protein knockdown results mirrored those for mRNA
knockdown obtained using real-time qRT-PCR (best knockdown observed
for 0.6 µl siPORT NeoFX conditions, data not shown).
Since cytotoxicity was a problem at the very highest amount
of transfection agent (1.2 µl/well), the best optimal transfection
conditions were determined to be 4,000 cells and 0.6 µl
siPORT NeoFX per well.
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Figure 1. Fast Transfection Optimization Using the Silencer® CellReady™ Transfection Optimization Kit and the KDalert™ GAPDH Assay Kit. 4,000 and 8,000 HepG2 cells were transfected with either Silencer® GAPDH or Negative Control #1 siRNA using a Silencer CellReady siRNA Transfection Optimization plate and varying amounts of siPORT™ NeoFX™ Transfection Agent. Silencing of GAPDH expression for each transfection condition was measured using the KDalert assay. Residual GAPDH activity was determined from the ratio of GAPDH activity in samples transfected with GAPDH siRNA divided by the GAPDH activity in corresponding samples transfected with Negative Control #1 siRNA. Transfection associated cytotoxicity for each of the transfection conditions was also measured using the KDalert GAPDH Assay Kit by comparing GAPDH activity of negative control siRNA transfected samples to that of untreated samples. Identified optimal transfection conditions are highlighted in pink. |
Optimized Transfection Conditions Can be Used
with Confidence
The optimal transfection conditions for HepG2
cells were used to transfect Ambion Silencer® Pre-designed
siRNAs targeting four additional genes (CDK2, STAT1, JAK1, and
PCNA). Two days after transfection the amount of gene knockdown
was measured for each transfected HepG2 sample using real-time
qRT-PCR (Figure 2). Each gene target was efficiently silenced
using the optimized transfection procedure. For these genes,
the optimized transfection conditions provided the highest levels
of target silencing compared with other tested transfection optimization
conditions (data not shown).
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Figure
2. KDalert™ Optimized
Transfection Conditions Silence Five Different Genes. HepG2
cells were transfected with Silencer® Pre-designed
siRNAs targeting five different genes in 96 well microplates
using the optimized conditions determined in Figure
1 (4,000 cells/well, 0.6 µl siPORT™ NeoFX™/well).
Two days after transfection, the mRNA levels in each
of the transfected cultures were compared to cultures
transfected with Negative Control #1 siRNA
using real-time qRT-PCR.
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Ambion's Complete Solution for siRNA Transfection
The KDalert assay is an excellent complement
to Ambion’s suite of control siRNAs and reagents for siRNA
transfection. The great ease and speed of the KDalert procedure
make it an ideal replacement for qRT-PCR based methods for transfection
optimization. For information on additional products that simplify
siRNA experiments, see Ambion’s RNAi Resource.
Scientific Contributors
Luis Foncerrada, Kevin Kelnar • Ambion,
Inc.
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