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RNA Interference
Complete Toolkit for Transfection
Optimization
• New Silencer® CellReady™ siRNA Transfection
Optimization Kit
• New KDalert™ GAPDH Assay Kit
• Silencer Positive and Negative Control siRNAs
• Silencer siRNA Transfection II Kit
Optimizing siRNA delivery and cell viability
during transfection assays eliminates the most common causes
of unsuccessful gene silencing experiments. Whether you will
be screening a new Silencer CellReady
siRNA Library (pre-plated siRNA ready for transfection) or one
of the larger Silencer siRNA Libraries (1–5 nmol siRNAs),
your experiments should start with optimization of transfection.
Ambion now offers a comprehensive set of reagents to facilitate
these experiments.
New! Silencer CellReady siRNA Transfection
Optimization Kit
Before using the new Silencer CellReady Libraries, it is
imperative to optimize transfection conditions for your cells
in 96 well plates. This can be done with ease using the Silencer CellReady
siRNA Transfection Optimization Kit. Developed specifically
for transfection optimization of human cell lines using the
same format as the CellReady Libraries, the Silencer CellReady
siRNA Transfection Optimization Kit includes three 96 well
plates containing positive (Silencer GAPDH siRNA)
and negative (Silencer Negative Control #1 siRNA)
control siRNAs, as well as 0.4 ml siPORT NeoFX and
detailed instructions for determining optimal transfection
parameters.
Silencer Transfection Kit II
In general, the first step when optimizing transfection conditions
for a new cell type is to test siRNA delivery efficiency using
different transfection agents and a positive and negative control
siRNA. The Silencer Transfection Kit II is the ideal
choice for optimizing siRNA transfection conditions in any size
plate. This kit, which can be used with human, mouse, and rat
cells, includes two different siRNA transfection agents—siPORT NeoFX and
siPORT Amine—as well as the highly validated Silencer GAPDH
siRNA and Silencer Negative Control #1 siRNA. siPORT Amine is
a polyamine mixture, whereas siPORT NeoFX consists of
a mixture of cationic and neutral lipids. These reagents support
efficient, reproducible siRNA transfections in different cell
types, and both have been validated for use with reverse transfection.
Silencer Positive and Negative
Control siRNAs
If you have already identified a desired transfection
agent, yet want to either fine tune transfection conditions or
to monitor transfection in your experiments, consider one of
the Silencer GAPDH
siRNAs (see sidebar, Why Use GAPDH Control siRNAs?).
Two GAPDH positive controls are fully validated for use in human
cells, and one is also optimized for mouse and rat cells. Silencer Control
siRNAs include 5 nmol of ready-to-use chemically synthesized
GAPDH siRNA, and both include a separate negative control siRNA
(2 nmol, Silencer Negative Control #1 siRNA).
Of course, every experiment requires a good negative control,
and most experts agree that the best negative control is a non-targeting
siRNA. Ambion offers three extensively tested and unique Negative
Control siRNAs that have no significant sequence similarity to
mouse, rat, or human gene sequences. Silencer Negative
Controls include 5 nmol of ready-to-use chemically synthesized
siRNA.
New! KDalert GAPDH Assay Kit
Ambion’s rapid fluorescence-based KDalert GAPDH Assay
Kit accurately measures GAPDH enzymatic activity in cultured
cells derived from human, mouse, and rat (for more details, see A
New Way to Assess siRNA Delivery Efficiency), making
it the perfect complement to the Silencer CellReady
siRNA Transfection Optimization Kit, the Silencer siRNA
Transfection II Kit, and the stand-alone Silencer GAPDH
siRNA Controls. The assay was developed especially for cells
transfected in 96 well plates, but is flexible enough for evaluation
of transfection in other plate formats as well. The KDalert GAPDH
Assay Kit protocol is quicker and easier than traditional
methods (e.g., Western blotting, quantitative RT-PCR) for measuring
siRNA-induced knockdown, greatly accelerating transfection optimization
experiments.
Optimal Conditions for Optimal Results
Investing the time to identify the best transfection
conditions for your experimental system will limit transfection
variability, and maximize data quality by maximizing siRNA uptake
and minimizing cellular toxicity. This is especially important
for 96 well format RNAi screening experiments, which require
a fairly significant expenditure of time and reagents. Once you
have identified an optimized transfection protocol, the interesting
and productive work of siRNA library screening can begin!
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