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Optimizing siRNA Transfection for RNAi
Low transfection efficiency and low cell viability
are the most frequent causes of unsuccessful gene silencing experiments.
Through careful optimization--e.g. choosing the right transfection
agent and transfection method--high levels of transfection efficiency
can be achieved in many cell types. Once a protocol is optimized
for a particular cell type, reproducible siRNA screening experiments
can easily be done.
Efficient Transfection is Critical
The ability of small interfering RNAs (siRNAs)
to silence gene expression is proving to be invaluable for studying
gene function in cultured mammalian cells. Quite often, the success
or failure of an siRNA experiment hinges on siRNA delivery. siRNAs
can be transiently transfected using commonly available transfection
reagents. However, high efficiency transfection of siRNA is not
trivial; therefore, the utility of RNAi in difficult-to-transfect
cell types may be limited.
To achieve maximum effectiveness of exogenously
introduced siRNAs, transfection optimization experiments are
required. Failure to optimize critical transfection parameters
can render RNAi effects undetectable in cell culture. These transfection
parameters include culture conditions, choice and amount of transfection
agent, exposure time of transfection agent to cells, and siRNA
quantity and quality. The transfection procedure itself can be
a critical factor. The pre-plated transfection procedure involves
pre-plating cells, meaning the cells are allowed to attach, recover,
and grow for 24 hours prior to transfection. Here, we show evidence
that an alternative transfection procedure, termed reverse transfection
[1] or neofection [2], offers several key benefits over the traditional
pre-plating method. Reverse transfection involves simultaneously
transfecting and plating cells, much like procedures used for
transfecting suspension cells. This method is easier and faster
because it bypasses several steps of the traditional procedure.
This article summarizes the use of reverse transfection to maximize
performance of siRNA in cultured cells and offers suggestions
on how to optimize siRNA transfection parameters.
Important Parameters in siRNA Transfection Experiments
• Health of cultured cells
• Transfection method
• Transfection conditions
• Quality and quantity of siRNA
The goal of transfection optimization
is to determine the conditions that will provide maximum gene
knockdown while maintaining an acceptable level of viability
for the particular cell type (see sidebar, Two-Step Optimization
Protocol).
Health of cultured cells.
For maximal cell viability during transfection, cells must be healthy at the
beginning of the experiment--healthy cells are easier to transfect than poorly
maintained cells. Overly crowded and sparse cultures are not conducive for
cell health. Many cells undergo expression profile changes that can adversely
affect your experiments when they are stressed by culture conditions. As a
rule, cells should never be allowed to cover the entire surface area of their
culture dish. Instead, cells should take up between 20 and 80 percent of the
available space. Subculturing cells before they become overcrowded minimizes
instability in continuous cell lines and reduces variability from experiment
to experiment. Cells can gradually change in culture, and it is difficult to
consistently maintain cells in perfect health; therefore, to obtain maximally
reproducible experimental results, we recommend that cells be transfected within
10 passages of the optimization experiments. Cells older than this should be
destroyed and replaced with new cells from a frozen stock. Finally, maintaining
strict protocols, including time intervals between plating and transfecting
cells, will improve experimental reproducibility.
Transfection method.
In preparation for transfection, adherent mammalian cells have been traditionally
pre-plated into tissue culture wells and allowed to attach, recover, and
grow for 24 h prior to transfection. Reverse transfection is an alternative
method of transfection where cells are transfected while still in suspension
(i.e. after trypsinization and prior to plating). The method produces equivalent
or improved transfection efficiency over the standard pre-plated method for
many of the cell types tested and saves an entire day in the process (Figure
1). Presumably, the amount of exposed cell surface, and not the number of
transfection complexes, is the limiting factor in traditional adherent transfection.
Reverse transfection is believed to increase cell exposure to transfection
complexes often leading to greater transfection efficiency.
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Figure 1. Standard
Pre-Plated Transfection vs. Reverse Transfection with
siPORT™ NeoFX™ Transfection Agent. Mammalian
adherent cells are typically "pre-plated" prior to
transfection, allowing them to reattach and resume
growth for 24 h before exposure to transfection complexes
(left). Reverse transfection or "neofection" involves
adding transfection complexes to the cells while they
are in suspension, prior to plating, thus saving an
entire day in the transfection procedure (right). |
Figure 2A shows reverse transfection of a GAPDH siRNA into
seven different mammalian cell types. The data suggests that
reverse transfection can deliver high levels of functional siRNA
to a wide variety of cells. Some cell types transfected more
efficiently by reverse transfection than the traditional method.
For example, HepG2 cells, traditionally a difficult cell line
to transfect, reverse transfected remarkably well (Figure 2B),
perhaps because their dense growth pattern precludes adequate
cell surface exposure to transfection agents once attached to
a substrate.
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Figure 2. Efficient
Reverse Transfection of Various Cell Lines. (A) Reverse
transfection of a GAPDH siRNA (Silencer® GAPDH
siRNA, Ambion) into seven different mammalian cell
types. Amounts of siPORT™ NeoFX™ (Ambion),
siRNA amounts, and cell density were optimized for
each cell line (data not shown). All cells were harvested
and analyzed by real-time RT-PCR for GAPDH mRNA levels
at 48 hours after transfection. (B) HepG2 and
HeLa cells were both reverse transfected during plating
and transfected after pre-plating the cells with an
siRNA targeting GAPDH (Silencer® GAPDH
siRNA, Ambion) or Negative Control siRNA (Silencer® Negative
Control #1 siRNA, Ambion). 48 hours post-transfection,
GAPDH expression was measured by real-time RT-PCR.
Percent gene expression was calculated as GAPDH gene
expression in GAPDH siRNA transfected cells compared
to those transfected with the Negative Control siRNA. |
Cell density became a less critical parameter, requiring little
to no optimization, when cells were reverse transfected. Figure
3A demonstrates that a broad range of cell concentrations were
reverse transfected efficiently, whereas traditional pre-plated
transfections required careful optimization of cell density (Figure
3B). In addition, reverse transfection is faster--a full day
can be saved because cells do not have to be plated prior to
transfection. Because of these fundamental advantages, Ambion
scientists routinely optimize transfection of new cell lines
using the reverse transfection procedure.
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Figure 3. Reverse
Transfection Yields Higher Tolerance to Cell Plating
Density. (A) SKOv3 cells were reverse transfected
in a 96 well plate using 10 nM and 30 nM GAPDH siRNA
(Silencer® GAPDH siRNA, Ambion)
or Negative Control siRNA (Silencer® Negative
Control #1 siRNA, Ambion) at the indicated cell densities
using siPORT™ NeoFX™ Transfection
Agent (0.3 µl per well, Ambion). At 48 hours post-transfection,
cells were harvested and analyzed by real-time RT-PCR
for both GAPDH mRNA and 18S rRNA levels. Remaining
gene expression was calculated as a percentage of GAPDH
mRNA in cells transfected with GAPDH siRNA compared
to cells transfected with Negative Control siRNA. Data
were normalized against the 18S rRNA signal. (B) COS-7
cells were pre-plated in a 24 well dish at the indicated
plating densities 24 hours prior to transfection. Transfections
were performed with either 10 nM GAPDH or Negative
Control #1 siRNA using siPORT™ Amine Transfection
Agent (4 µl per well, Ambion). Remaining gene
expression was determined as described for Panel A.
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Transfection conditions.
Overall, transfection
efficiency and cell viability are dependent on choice and amount
of transfection agent and exposure time of cells to transfection
agent. Commercially available reagents perform with varying levels
of effectiveness depending on the cell type. A successful match
between cell line and reagent can usually be made by testing
several commercially available agents. Transfection agent volume
is also critical--too little will not transfect efficiently;
too much can be cytotoxic. Both siRNA transfection efficiency
and cell viability should be considered when designing transfection
agent screening experiments. The ideal reagent is one that yields
effective target gene reduction without significant cell mortality.
In HepG2 cells, siPORT™ NeoFX™ Transfection
Agent shows minimal toxicity and yields a broad range of silencing
activity (Figure 4). Some reagents and cell lines are not as
flexible and require more precision. Ambion recommends testing
transfection agents that have been validated specifically for
siRNA transfection. We have found that most DNA-based transfection
agents are ineffective for siRNA delivery (see Ambion's
recommendations).
| |
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Figure 4. Transfection
Agent Cytotoxicity. Multiple siPORT™ NeoFX™ Transfection
Agent volumes (0.03-1.0 µl, Ambion) were
used in reverse transfection of HepG2 cells. Assays
were done in 96 well plates with 5 nM GAPDH siRNA (Silencer® GAPDH
siRNA, Ambion) or Negative Control siRNA (Silencer® Negative
Control #1 siRNA, Ambion). Remaining gene expression
(bars) was determined as described for Figure 3A. Cell
viability (line) was also measured using the ViaCount® Assay
(Guava Technologies). As reagent volume increased,
greater levels of siRNA-mediated reduction of target
gene expression were obtained. In this cell type, siPORT NeoFX shows
minimal toxicity and yields a broad range of silencing
activity. Some reagents are not as flexible and require
more precision. |
Length of cell exposure to transfection agents
should be optimized to minimize cellular toxicity and maximize
siRNA activity by varying the amount of transfection agent
and cell exposure time to transfection complexes (Figure 5).
Media containing transfection agent was removed from the wells
at the indicated time points and replaced with fresh media. Cellular
viability, apoptosis, and siRNA activity were measured 48 hours
after addition of 1 or 2 µl transfection agent + GAPDH siRNA
or negative control siRNA. In wells where transfection agent
was not removed, cells appeared necrotic, they underwent moderate
levels of apoptosis, and cell viability was >30% less than
a nontreated control well. These same samples, however, showed >90%
reduction in GAPDH gene expression over negative control wells.
When transfection complexes were removed at
4 hours post transfection, cell viability was >95%, apoptosis was minimal,
but GAPDH silencing was 30% less than in cultures experiencing no media change.
When cells were exposed to 1 µl transfection agent for 24 hours post-transfection
before a media change, GAPDH silencing was high, comparable to cells with
no media change, and cell viability was nearly 90%. These data suggest that
careful optimization of cell exposure to transfection complexes can improve
the quality of data generated in RNAi experiments.
 |
Figure 5. Exposure
Time to Transfection Complexes. HeLa cells (5 x
103 cells/well) were exposed to transfection
complexes containing one of two concentrations of transfection
agent (1-2 µl) + 10 nM GAPDH (Silencer® GAPDH
siRNA, Ambion) or negative control siRNA (Silencer® Negative
Control #1 siRNA, Ambion). Medium was changed to remove
transfection complexes at different time points. Cellular
viability, apoptosis, and siRNA activity were measured
48 h after transfection began. Cell viability (blue
line) was measured using the ViaCount® Assay
(Guava Technologies). Apoptosis (yellow line) was measured
using a Guava® PCA™-96 instrument
(Guava Technologies). Remaining gene expression (green
bars) was determined as described for Figure 3A. NT
= Not Transfected |
Quality and quantity of siRNA.
The quality
and quantity of siRNA used for transfection significantly influences
RNAi experiments. siRNA should be free of contaminants carried
over from synthesis including salts, proteins, and ethanol. Additionally,
the siRNA should also be <30 bp, because the presence of dsRNA
larger than approximately 30 bp has been shown to alter gene
expression by activating the nonspecific interferon response
[3].
The optimal concentration of siRNA is influenced
by several factors including properties of the target gene and
cell type. As mentioned above, too much siRNA may lead to off-target
effects; too little can result in undetectable gene silencing.
In general, 1-30 nM siRNA is a good concentration range within
which to optimize transfection (10 nM is a sufficient starting
point). In Figure 6, transfection of HeLa cells was optimized
at very low concentrations of siRNA. HeLa cells are easier to
transfect than many other cell types, and 10 nM siRNA in combination with reverse
transfection is sufficient for obtaining optimal target gene reduction.
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| Figure 6. Optimal
Amount of siRNA. HeLa cells were split and resuspended
in growth media at 4.0x104 cells/ml. Transfection
complexes were prepared containing the indicated concentration
of chemically synthesized GAPDH siRNA (Ambion) or Negative
Control siRNA #1 (Ambion; data not shown), and 0.3 µl
siPORT™ NeoFX™ Transfection Agent (Ambion).
48 hours post-transfection, cells were harvested and
analyzed by real-time RT-PCR for both GAPDH mRNA and
18S rRNA levels. Remaining gene expression was determined
as described for Figure 3A. |
Conclusion
There is no single transfection parameter
that by itself ensures efficient siRNA uptake by cells in culture.
Optimal siRNA uptake into viable cells is achieved by systematically
addressing each of several critical variables. Ambion provides
a series of tools to simplify RNAi experiments including two
transfection agents designed specifically for siRNA delivery.
Both siPORT NeoFX and siPORT Amine Transfection
Agents can be used for reverse transfection or standard transfection
of siRNAs into a wide variety of cell types. The siPORT
Transfection II Kit contains samples of both of these reagents as well as
positive and negative control siRNAs for protocol optimization.
Ambion's siRNA Delivery Resource has further details on
transfection optimization, recommendations for transfection agent,
and conditions to use with specific cell types.
siPORT™ Amine is manufactured
for Ambion by Mirus.
Scientific Contributors
Rich Jarvis • Ambion, Inc.
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