|
Silencer® siRNA
Starter Kit
New User's Kit for Gene Silencing
Just starting siRNA experiments? The new Silencer® siRNA
Starter Kit is perfect for researchers new to RNAi. It provides
all of the reagents, protocols, and instructions necessary to
help you get underway with siRNA transfection and to ensure that
your gene knockdown experiments are successful.
The Silencer siRNA Starter Kit includes
a high quality, lipid-based transfection agent, siPORT™ NeoFX™,
that effectively delivers siRNAs to a broad range of cell lines.
siPORT NeoFX can be used with reverse transfection,
a streamlined protocol that cuts out an entire day from the RNAi
experimental procedure; it can also be used with standard transfection
protocols. This reagent is not sensitive to serum and efficiently
transfects low concentrations of siRNA.
Positive and negative control siRNAs are included
for siRNA delivery optimization as well as knockdown assessment.
The positive control, a well characterized GAPDH siRNA, has been
shown to strongly down regulate GADPH in human, mouse, and rat
cells (Figure 1).
|
|
| Figure
1. Silencing of GAPDH
Expression in Human Lung Carcinoma (A549), Rat Osteosarcoma
(UMR106), and Mouse Fibroblast (3T3) Cells Using the Silencer® siRNA
Starter Kit. Each cell line was transfected using
siPORT™ NeoFX™ Transfection Agent.
(A) Measurement of siRNA mediated silencing of GAPDH expression
in three mammalian cell lines using the KDalert™ GAPDH
Assay. Silencer GAPDH or Negative Control #1 siRNA
was reverse transfected into cells in 96 well plates (4,000
cells per well) in duplicate using the recommended protocol
provided in the kit. Two days after transfection the cells
were harvested, and the amount of GAPDH enzymatic activity
in each sample was measured using the KDalert GAPDH Enzyme
Assay supplied with the kit. Reduced GAPDH is observed
in samples from cultures transfected with GAPDH siRNA compared
to cultures transfected with Negative Control #1. (B) Detection
of specific GAPDH mRNA knockdown in A549 cultures transfected
in a 96 well plate with GAPDH siRNA detected by real-time
qRT-PCR analysis. GAPDH mRNA in samples derived from GAPDH
siRNA-transfected cultures is detected at a higher Ct than
in samples derived from cultures transfected with Silencer Negative
Control #1 siRNA. The inset shows the relative reduction
in GAPDH mRNA levels for cultures transfected with GAPDH
siRNA versus that of the negative control transfected cells.
(C) Western blot analysis of the specific reduction in
GAPDH protein expression in siRNA-transfected A549 cells.
5 µg of total protein was loaded in each well. In
both Silencer Negative Control #1- and GAPDH siRNA-transfected samples, a specific 36 kDa immunoreactive band
is detected using the antibody supplied in the kit. Cells
transfected with GAPDH siRNA show a lower expression level
of GAPDH protein compared to cells that were treated with Silencer Negative
Control #1 siRNA. |
An anti-GAPDH antibody is provided to assess
GAPDH knockdown by Western blot or immunofluorescence. Ambion’s
KDalert™ GAPDH Assay is also provided to quickly assess
GAPDH enzymatic activity (See Quickly Assess siRNA Delivery and
Cell Viability in the Same Assay). This assay has
the added benefit that it can be used to assess cell viability.
Finally, RT-PCR primers, for real-time quantitative RT-PCR detection
of GAPDH mRNA using SYBR® Green,
are included.
This complete set of reagents makes it possible
to perform and optimize siRNA transfection experiments. These
reagents can also be used to transfect miRNAs, such as Ambion’s
Pre-miR™ miRNA Precursor Molecules. Stand alone components can
be reordered as necessary.
Scientific Contributors
Luis Foncerrada, Kevin Kelnar, Lesslie Beauchamp,
Lance Ford,
Joe Krebs • Ambion, Inc.
|
