siRNA transfection optimization
with the Agilent 2100 bioanalyzer
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When working in the field of target validation, or when studying functional
assays requiring the transfection of small interfering RNA (siRNA)
into a cell, the presence of having a fast, easy-to-use method to optimize
transfection conditions for given cell lines would be of great value.
Three to four siRNA sequences are generally tested for each gene in
order to suppress the expression of a protein with a corresponding
mRNA sequence. Before any sequence comparison can be performed,
the integrity and purity of siRNA, the siRNA uptake and the cell viability
must be monitored and optimized. Once optimal transfection conditions
have been established, different siRNA sequences can be evaluated for
the lower target protein expression. There are many influencing factors
for transfection efficiency, among them the cell line, siRNA concentration
and its ratio to the transfection reagent, cell confluence during transfection,
incubation time and media composition all need to be taken into
consideration. In this Application Note we describe the use of Agilent’s
2100 bioanalyzer to quickly verify the siRNA integrity and to determine the
optimal transfection conditions for gene silencing experiments with the
help of red-fluorescently labeled siRNA and cell fluorescence assays.
Cell viability and delivery efficacy are simultaneously evaluated with
fluorescent probes, therefore helping to decide on the optimal transfection
conditions.
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Over recent years the scientific
community has seen an explosion
in the number of research studies
related to RNA interference
(RNAi) induced gene silencing.
Benefits, such as genome usability
to generate knockout phenotypes,
easy automation with the potential
for high throughput, specificity,
and low cost when compared to
animal knockout models are seen
as a huge opportunity for functional
analysis, target validation
and gene-specific therapeutics.
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RNAi is a well-conserved cellular
mechanism believed to have antiretroviral
effects. dsRNA fragments
with homologous sequence to a
translated mRNA fragment will
effectively silence the expression
of the protein by catalyzing the
degradation of the complementary
mRNA1-4. In RNAi Dicer, an RNase
like enzyme, is responsible for
processing dsRNA into double
stranded small interfering RNA
(siRNA). In vitro studies in
Drosophila suggested that siRNAs
assemble into endoribonuclease
enzyme complexes known as
RNA-induced silencing complexes
(RISCs). After the siRNA strands
are unwound, activated RISCs are
guided to complementary mRNA
molecules, where they cleave the
associated RNA (for recent
reviews, Refs. 5-6).
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While in some organisms, such as
C. elegans or Arabidopsis, RNAi
requires an RNA amplification step7,
the direct introduction of siRNA in
mammalian cells has shown to
produce transient silencing of more
than 90 % in protein expression.
Recovery from a single treatment occurs after 4 to 6 days8,
therefore suggesting that no
copying takes place.
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There are currently several
methods for siRNA delivery into
cells. siRNA can be produced in
vitro by chemical synthesis, in
vitro transcription or RNase III-type
digestion of dsRNA or siRNA can
be directly expressed in vivo by
use of plasmids, viral vectors or a
PCR cassette. All of these methods
require the optimization of the
transfection, for cell viability, siRNA
uptake/production and silencing
effectiveness. It is known that
optimal transfection conditions
strongly vary between cell lines.
However, transfection is not
affected by the specific sequence
of the nucleic acid or the presence
of a fluorescence tag, which
allows the use of the optimized
conditions for the screening of
several sequences. Also, in vitro
preparation requires additional
purification steps where the
verification of purity and integrity
of nucleic acids is required.
Given the expense and complexity
of monitoring and optimizing
these types of experiments, a new
tool that allows for minimal sample
and reagent consumption in a fast
and automated format would be of
great benefit.
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The Agilent 2100 bioanalyzer
combines electrophoretic analysis
of nucleic acids with dual-color
flow cytometry capabilities. The
DNA 1000 LabChip kit can be
used for verifying purity and
integrity of dsRNA from siRNAs
(19-23 bp) up to 1000 bp dsRNAs
while the Cell Fluorescence
LabChip® kit is the ideal tool for
monitoring transfection efficiency of fluorescently tagged siRNAs or
green fluorescent protein (GFP)
producing vectors. Compared to
fluorescence microscopy, the
Agilent 2100 bioanalyzer automates
the measurements, increasing the
total cell count to approximately a
thousand cells per sample and
provides quantitative information
on the transfection degree and
viability of each cell. In addition,
the silencing effect of RNAi can be
quickly monitored with the Agilent
2100 bioanalyzer by antibody
staining of cells or analysis of
endpoint RT-PCR results. A typical
workflow for a siRNA transfection
optimization experiment is outlined
in figure 1, showing analysis time
reduced to 60 minutes. We show
data on electrophoretic quality
assessment of siRNA and transfection
efficiency measurements
of red fluorescence tagged siRNA.
Varying amounts of siRNA, different
transfection reagents and its ratios
were evaluated.
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Materials and Methods
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Short interfering RNA preparation
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Cy5® labeled and unlabeled short
interfering RNA were synthesized
by QIAGEN. For the annealing
of siRNA duplexes, 5 nmol single
stranded sequences targeting
Lamin A/C (QIAGEN, Cat. #:
1022050), were incubated in 250 µl
siRNA Buffer (QIAGEN) for one min
at 91 °C followed by 1 h at 37 °C.
GFP siRNA (QIAGEN, GFP-22
siRNA, sense 3’Cy5 modified sense
and antisense 5’-P(Phosphate))
was supplied annealed and ready
to use in sterile buffer. Samples
were then stored at –20 °C until
needed, and then incubated at 37 °C
for 20 min.
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Electrophoretic measurement
of purity and integrity
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dsRNA electrophoresis analysis
was performed using the DNA 1000
LabChip kit with the Agilent 2100
bioanalyzer. The kit includes dsDNA
internal standards at 15 and 1500 bp
for sample alignment (sizing) and
quantitation. Lamin A/C siRNA
was analyzed after 1:10 dilution in
PBS.
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Transfection and culture conditions
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Lipid based TransMessenger™
and RNAiFect™ transfection
reagents where kindly provided by
QIAGEN GmbH. Human cervix
carcinoma Hela S3 cells (DSMZ,
ACC 161) were kept in culture with
10 % FBS in Ham’s F12 medium
at 5 % CO2 atmosphere and 37 °C.
Twenty-four hours before transfection,
5x104 cells where seeded in
24 well plates. Transfection protocol
was followed as described in QIAGEN’s manual for each transfection
reagent, being the specific
siRNA incubations times of 3 hours
in serum free medium for Trans-
Messenger and 17 hours in complete
medium when RNAiFect was used.
After transfection, cells were kept
in culture medium until time of
analysis, 24 hours after transfection
was initiated.
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Transfection efficiency measurements
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Flow cytometry measurements
were performed using the Cell
Fluorescence LabChip kit with the
Agilent 2100 bioanalyzer. After
enzymatic cell detachment with
Accutase (PAA laboratories, Cat. #
L11-007) cells where resuspended
in Cell Buffer at a concentration of
2x106 cells/ml. For on-chip staining
procedure, cells were loaded onto
a cell chip (10µl/sample), vortexed
for 1 min (1000 rpm) and incubated
for 20 min in the dark with 0.5µl of
the life staining dye CellTracker
Green CMFDA (5-chloromethylfluorescein
diacetate, Molecular
Probes, Cat. # C-7025) at a final
concentration of 6 µM. After 1 min
vortexing (1000 rpm) the chip
was loaded into the instrument
and the six samples were analyzed
automatically.
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Results and Discussion
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The first step of an RNAi
experiment is the assessment
of purity and integrity of the
interfering RNA. Figure 2 shows
the electropherogram and gel-like
image of a chemically synthesized
siRNA. The size range and resolution
of the DNA 1000 assay match the
requirements for siRNA quality
control, verifying that a single
sharp peak for siRNA is obtained
and undercovering any impurities
ranging 15 to 1000 bp coming from
digestion, synthesis, purification
or degradation products. In case
of RNAse III or Dicer digestion
of dsRNA the appearance of
fragments above 30 bp would
indicate incomplete digestion and
potentially induce the interferon
mediated unspecific suppression
of gene expression in mammalian
cells10.
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It is also possible to evaluate
RNAse activity of culture medium
with serum as the use of serum can
increase transfection efficiency in
some cell lines, this does however
add to the risk of RNA degradation.
siRNA is much more resistant to
nuclease degradation, although
long term incubation in serum
containing medium can effectively
degrade it. Following siRNA
integrity validation, cells were
transfected as described in the
methods section. Two different
lipid based transfection reagents
were evaluated, cell confluence
at time of transfection was kept
constant at about 75 % for
TransMessenger and 50 % for
RNAiFect. Optimal conditions
were determined by reaching the
maximum of Transfection Viability
(TV)(equation 1). This factor, as
defined, combines two important
parameters for determining the
success of any transfection
experiment: transfection efficiency
(TE) and cell viability in transfected
cells (ViT). Transfection Viability
is calculated as the product of the
percentage of transfected cells in
the live cell population (transfection
efficiency) and the ratio of live
cells in the transfected population
(figure 3). When evaluating the
Transfection Viability with Trans-
Messenger transfection reagent, we
measured the effect of increasing
the siRNA to TransMessenger
ratio while keeping the siRNA
amount fixed (0.4 µg/sample).
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In this experiment, increasing
amounts of TransMessenger steadily
increased the transfection efficiency
while gradually decreasing the
viability of the transfected cells
(figure 4A). The relatively low
amount of siRNA suggests that the
absolute amount of transfection
reagent was also low, allowing for
higher ratios of TransMessenger
without a direct effect on viability.
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The effect of changing the siRNA
amount while keeping a fix ratio
(1:4) was more pronounced
(figure 4B). A maximum for
Transfection Viability is observed
at 0.4 µg siRNA/sample; it quickly
drops with decreasing or increasing
amounts due to too low siRNA
concentration or toxicity when in
excess.
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The Transfection Viability with
RNAiFect was evaluated as
suggested by QIAGEN protocols
for Hela cells, in the range from
0.5 to 1.5 µg siRNA/sample in a 24
well plate and siRNA to RNAiFect
ratios from 1:3 to 1:9. It shows very
high Transfection Viability in Hela
cells for most conditions. The effect
of increasing amounts of siRNA
when compared to different siRNA
to RNAiFect ratios (figure 5) show
that with RNAiFect lower amounts
of siRNA could be successfully
used with higher ratios to produce
a good transfection, allowing cost
per analysis to be included in the
optimization criteria. The overall
TV maximum appears at 0.9 µg
siRNA with 5.4 µl RNAiFect (1:6),
showing the highest efficiency
with very high viability of the
transfected cells.
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Conclusion
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The Agilent 2100 bioanalyzer is
the ideal tool for fast and easy
optimization of transfection in
RNAi experiments. For successful
gene silencing it is essential to
optimize transfection conditions
for each cell line used, as siRNA
delivery into cells is one of the
most important steps. Transfection
success is improved by the inclusion
of viability of the transfected cells
in its definition. The microfluidic
system combines assays for
transfection viability measurement, cellular protein expression analysis
and siRNA integrity as well as
purity on a single platform. The
additional advantage that only a few
cells and mimimum reagents are
needed for optimization experiments
with the 2100 bioanalyzer means
that the remaining cells can be used
for further analysis. QIAGEN’s
ability to deliver an integrated
solution for RNAi reagents
complements the Agilent 2100
bioanalyzer and provides a new
analysis tool for effective gene
silencing.
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