NEW
Streamline Your siRNA Transfections
siPORT™ NeoFX™ Transfection Agent
Promotes High Reproducibility and Efficiency
• Fast--transfect cells as they
are plated (reverse transfection), saving a day •
Reproducible--produces consistent results, lot-to-lot,
plate-to-plate, and well-to-well • Versatile--works
with a broad range of cell lines •
Efficient--performs with high siRNA transfection efficiency,
allowing use of low siRNA concentrations to minimize
nonspecific effects • Powerful--performs with low
cytotoxicity in the presence or absence of serum
Streamlined Transfection Protocol
siRNA transfection is traditionally a two-day procedure,
making optimization a relatively slow process. siPORT NeoFX Transfection
Agent was developed to refine siRNA transfection protocols. siPORT NeoFX
lipid-based formulation can be used to efficiently transfect adherent
cells just after trypsinization--without increased cytotoxicity. Just
add siPORT NeoFX to your diluted siRNA, incubate to form transfection
complexes, add the complexes to the culture wells, and overlay with cells
(Figure 1). This new streamlined protocol, named "neofection," will save
researchers a full day of valuable time when compared to traditional pre-plated
transfection procedures. The transfection complexes are active and stable
even in the presence of serum, so there is no need to remove or replace
media following transfection. From start to finish, successful gene silencing
experiments can be completed in less than 24 hours. This streamlined protocol
can be adapted to a wide range of cells and experimental designs, including
high-throughput applications.
Figure 1. Traditional Plated Transfection vs.
Neofection with siPORT™ NeoFX™
Optimized for Lower siRNA Concentrations
Recent reports indicate that siRNA concentrations of 100
nM or higher can lead to nonspecific changes in gene expression (off-target
effects) in mammalian cultured cells [1-3]. Reducing the amount of siRNA
used for transfections to 1-20 nM minimizes these nonspecific effects,
while still providing extensive silencing of the target gene [3]. Ambion
scientists specifically developed the siPORT NeoFX Transfection
Agent for efficient transfection of low concentrations of siRNA. For example,
high levels of siPORT NeoFX-mediated gene silencing (>80%) were
achieved by transfection of as little as 0.1 nM siRNA (targeting GAPDH)
into HeLa cells (Figure 2). Using 3 and 10 nM siRNA (Silencer™ GAPDH
siRNA and Kinase
Library siRNAs) in siPORT NeoFX transfection complexes, substantial
reduction of target gene expression was observed in many adherent, human
cell lines, including MCF-7 (breast adenocarcinoma cells), SKOV3 (ovarian
cancer cells), A549 (lung carcinoma cells), SKNAS (bone marrow neuroblastoma
cells), and BJ cells (primary foreskin fibroblast cells) (data not shown).
Figure 2. Efficient
Transfection with Low siRNA Concentration. HeLa cells were trypsinized and resuspended
in growth media at a concentration of 4x104 cells/ml. Transfection
complexes were prepared using chemically synthesized GAPDH siRNA (0.03
nM to 10 nM) or negative control siRNA (data not shown) and 0.5 µl siPORT™
NeoFX™ Transfection Agent. 48 hours after transfection, cells were
harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S
rRNA levels. Percent mRNA remaining was calculated as the amount of GAPDH
mRNA in cells transfected with GAPDH siRNA compared to that of cells transfected
with the negative control siRNA. Data were normalized to the 18S rRNA
signal. These results demonstrate that siPORT NeoFX performs well
even with reduced levels of siRNA, which may offer significant benefits
for eliminating off-target effects in gene-silencing applications.
High siRNA Transfection Efficiency with Low
Cytotoxicity
siPORT NeoFX can achieve a high level of siRNA
transfection and knockdown efficiency while maintaining a high level of
cell viability. In a representative transfection experiment, Cy™3-labeled
siRNA was successfully delivered into a high percentage of HeLa cells
(Figure 3A). Furthermore, in comparison with other commercially available
transfection agents, siPORT NeoFX maintained the highest level
of cell viability while exhibiting excellent gene silencing activity in
HepG2 cells (Figure 3B).
Figure 3. High
Efficiency and Low Cytotoxicity of siPORT ™ NeoFX™ Transfection
Agent. (A) HeLa cells were transfected with 30 nM Cy™3-labeled siRNA
using siPORT NeoFX Transfection Agent. Cells were subsequently
fixed, stained with DAPI for visualization of nuclei, and analyzed by
fluorescence microscopy. Blue: DAPI stained nuclei; Red: Cy3 fluorescent
siRNA. (B) CDK2 siRNA or Silencer™ Negative Control siRNA
#1 was complexed with six different transfection agents according to manufacturer's
instructions and added to empty wells of a 96 well dish (final siRNA concentration
of 30 nM). HepG2 cells were added to the wells and cultured for 48 hours
in complete media containing serum. Cell viability was analyzed by microflow
cytometry using PCA 96 instrument and Viacount™ assay (Guava Technologies).
RNA was isolated, and target gene expression was quantified by real time
RT-PCR. Relative reduction in mRNA levels is expressed as a percentage
of expression in cells transfected with a negative control siRNA. Duplicate
samples were normalized by measuring 18S rRNA levels by real time RT-PCR.
The range of normalized gene expression is shown with error bars.
Lot-to-lot, Day-to-day, Plate-to-plate, and
Well-to-well Consistency in Performance
In addition to efficient siRNA transfection with low cytotoxicity,
a good transfection agent is also expected to reliably deliver high performance
among multiple wells for replicate samples and between different lots
of the transfection agent. Such characteristics are especially important
in high throughput screening of siRNA libraries arrayed in 96 well plates.
As shown in Figure 4, there was negligible variability in gene silencing
efficiency between multiple wells and multiple plates when siPORT NeoFX
was used in experiments on two different days. Further, three different
lots of siPORT NeoFX were found to give consistent knockdown efficiency
(data not shown).
Figure 4. Consistent
Day-to-day, Plate-to-plate, and Well-to-well Performance.
8,000 HeLa cells were transfected in 96 well plates using siPORT™ NeoFX™
(0.3 µl/100 µl reaction volume) and either GAPDH siRNA (10 nM) or a scrambled
negative control siRNA. Remaining GAPDH expression was quantified as described
in Figure 2. Each bar represents the mean of eight replicate wells. siPORT
NeoFX demonstrates consistent transfection performance across multiple
wells, among multiple plates, and on multiple days.
Solutions for siRNA Delivery
Although the RNAi field is still young, the
use of RNAi has already been demonstrated to have enormous
potential for understanding gene function, elucidating
biological pathways, and identifying and validating potential
drug targets. Ambion is committed to bringing researchers the
best solutions for siRNA delivery. Ambion's line of
transfection and electroporation products offers consistent
and reliable performance. siPORT NeoFX Transfection
Agent is unique in that it can be used during seeding of cells
into culture dishes, thereby streamlining the transfection
protocol and shortening the procedure by an entire day.
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Ordering Information
Cat#
Product Name
Size
1632
Silencer® siRNA Labeling Kit - Cy™3
65 µg labeled siRNA
4510
siPORT™ NeoFX™
Transfection Agent
0.4 ml
4511
siPORT™ NeoFX™
Transfection Agent
1 ml
4611
Silencer® Negative Control #1 siRNA
5 nmol (50 µM)