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Matched siRNAs and Assays
Ambion + Applied Biosystems =
RNAi Success
Ambion and Applied Biosystems have
joined forces to provide a complete convenient, solution for
performing gene silencing experiments and validating the results
by real-time RT-PCR. Ambion's Silencer™ Validated
and Silencer Pre-designed siRNAs eliminate the guesswork--and
the tedious labwork--associated with siRNA design and testing.
Applied Biosystems' gene-specific, ready-to-run TaqMan® Gene
Expression Assays provide you with a fast, simple way to measure
the efficacy of mRNA knockdown.
The ultimate goal of siRNA experiments
is to assign gene function, analyze biological pathways, or
validate potential drug targets. But before that goal can be
realized, the siRNA has to be designed, synthesized, delivered
to cells using an optimized protocol, and proven effective.
Finally, it is necessary to correlate any induced phenotypic
change with the extent of knockdown induced by the siRNA.
Ambion's Silencer™ Pre-designed
siRNAs for >34,000 human, mouse, and rat genes in the NCBI
RefSeq database eliminate costly and time-consuming siRNA design,
synthesis, and testing steps. Similarly, Applied Biosystems
offers TaqMan® Gene Expression Assays for quantitative
gene expression analysis of >41,000 human, mouse, and rat
genes by real-time PCR. These assays can be used to measure
mRNA knockdown for siRNA validation, to optimize transfection,
and to correlate phenotype with the extent of knockdown induced
by a particular siRNA.
Assays to Monitor Gene-specific siRNA Effects
siRNAs exert their effects at the mRNA
level. Therefore, the preferred assay for siRNA validation
and transfection optimization purposes is one that quantitates
target mRNA levels. Once these preliminary studies are complete,
there is an advantage to measuring both target mRNA and corresponding
protein levels to correlate phenotypic changes--monitored by
enzymatic assay, cell based assay, gene expression profiling,
or other means--with extent of knockdown induced by an siRNA.
Advantages of TaqMan Gene Expression Assays
qRT-PCR provides several advantages
for monitoring target mRNA levels in RNAi experiments. It is
thousands of times more sensitive than Northern analysis, results
can be obtained much more quickly (assays complete in only
a few hours), and the method provides quantitative results.
When used with Applied Biosystems TaqMan Gene Expression Assays--off-the-shelf,
pre-designed and pre-optimized primer-probe sets available
for >21,000 human, >14,000 mouse, and >4,000 rat genes--the
method requires no optimization. This makes real-time PCR easier
to perform and the data obtained more reproducible with less
signal variance than Northern analysis.
siRNA Validation
Gene silencing experiments require siRNAs
that efficiently knock down expression of the target gene.
To prove that a particular siRNA sequence is effective, the
siRNA needs to be functionally tested in cells and be proven,
or "validated," to reduce target mRNA levels by a predetermined
amount. Functional testing of a large number of siRNAs designed
using a particular siRNA design algorithm is also necessary
to prove that the algorithm used to design the siRNA accurately
predicts effective siRNAs.
Ambion, and partner Cenix BioScience,
chose qRT-PCR to test Ambion's Silencer Validated
siRNAs and to systematically validate more than 1100 siRNAs to verify
the effectiveness of Cenix's siRNA design algorithm (this algorithm
was used to design all of Ambion's Silencer Validated
and Pre-designed siRNAs; see siRNA Design: It's All in the Algorithm). Figure 1 illustrates the workflow
used to validate siRNAs. Using this method, siRNAs that are
determined to reduce their target mRNA level by 70% or more
are considered "validated" and
are subsequently made available as Silencer Validated
siRNAs. Figure 2 shows a small subset of validation data generated
at Ambion using TaqMan Gene Expression Assays to quantitate
target mRNA levels.
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Figure 1. Validation
of Ambion's Silencer™ Validated
siRNAs. The following
procedure is used by both Cenix and Ambion to validate
siRNAs:
1) Cells are plated in 96
well plates and grown for 24 hours.
2) Gene specific and negative control siRNAs
are independently transfected in triplicate.
3) 48 hours later, RNA is extracted.
4) Target mRNA levels are quantitated by real-time
PCR.
5) Data are normalized using 18S rRNA levels.
6) The extent of target gene knockdown is
expressed as a percent of mRNA remaining in cells
treated with the gene-specific siRNA
compared to cells treated with a negative control siRNA (Silencer Negative
Control siRNA #1).
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Figure 2. Silencer ™ siRNA
Validation Data Generated Using Applied Biosystems
TaqMan® Gene Expression Assays. The
indicated Silencer Validated
siRNAs were transfected into HeLa Cells at 30 nM. RNA
was isolated 48 hours later and analyzed by one-step
qRT-PCR using the appropriate TaqMan Gene Expression
Assay (results were normalized for input RNA amount
using real-time data for 18S rRNA). The inset graphs
show the reduction in target gene expression compared
to cells transfected with an equal concentration of Silencer Negative
Control #1.
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Optimizing siRNA Transfection
Before siRNA experiments can be conducted
to study gene function, analyze biological pathways, or validate
drug targets, optimized siRNA delivery conditions must be identified
and/or verified. The best way to test, optimize, and validate
siRNA transfection or electroporation conditions is to use
a highly effective, verified siRNA to an appropriate target
and to monitor decreases in mRNA levels induced by delivery
of the siRNA. Ambion uses qRT-PCR as the method of choice,
and Applied Biosystems TaqMan Gene Expression Assays provide
validated, easy-to-use primer-probe sets for this purpose (Figures
3 and 4).
The Silencer GAPDH
siRNA Control--and
the TaqMan Gene Expression Assay for GAPDH--provide the ideal
control siRNA and assay for optimization of transfection, respectively,
(Figure 4).
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Figure 3. Overview
of the Process for siRNA Transfection Optimization.
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Figure 4. Using
qRT-PCR to Optimize Transfection of siRNA. Cos-7
cells were transfected with CDK2 siRNA or a negative
control siRNA using the indicated volumes of transfection
agent per well. 48 hr after transfection, the cells
were harvested and analyzed by real-time RT-PCR using
TaqMan® Gene Expression Assays for CDK2
and 18S rRNA. Data from the 18S rRNA reactions were
used to normalize input RNA, and the percent CDK2 expression
was calculated as the amount of gene expression compared
to the negative control siRNA. |
Correlating Phenotype with the Extent of
Knockdown
To better evaluate functional siRNA
experimental results, the phenotype elicited should be correlated
with the extent of knockdown induced by a particular siRNA.
For a complete picture, both target mRNA levels and corresponding
protein levels should be analyzed. Protein levels can be monitored
by Western blot, immunofluorescence, ELISA or other means.
(Protein can be isolated using Ambion's PARIS™ Kit.)
For quantitating mRNA levels, however, qRT-PCR is again the
preferred choice.
An example in which the phenotypic effects
of an siRNA were correlated with the extent of mRNA knockdown
induced is shown in Figure 5. A Silencer Validated siRNA
targeting survivin mRNA was transfected into cells. Silencer Negative
Control #1 siRNA was transfected into another set of cells.
qRT-PCR using a TaqMan Gene Expression Assay showed that the
survivin siRNA reduced survivin mRNA levels in these cells
by 80% compared to cells treated with the negative control
siRNA (Figure 5). In addition, immunofluorescence analysis
of survivin protein levels indicated that protein levels were
reduced 76% compared to cells transfected with the negative
control siRNA (data not shown). In the survivin siRNA treated
samples, distinct changes in nuclear morphology, consistent
with changes that would be expected for cells undergoing apoptosis,
were noted. No distinguishable change in nuclear morphology
was noted in cells treated with the negative control siRNA
as compared to nontransfected cells. From these data, it can
be inferred that knockdown of survivin induces apoptosis in
these samples. The next experimental step would be to confirm
the results with a second siRNA targeting survivin, and to
monitor apoptosis by additional assays (e.g., caspase activity
assay, annexin V assay, etc.), while continuing to monitor
the extent of survivin mRNA knockdown by qRT-PCR.
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Figure 5. Knockdown
of Survivin with a Silencer™ Validated
siRNA and Verification with TaqMan® Gene
Expression Assays. HeLa
cells were transfected with 30 nM of a Silencer Validated
siRNA targeting survivin (BIRC5) or Silencer Negative
Control #1 in quadruplicate. Forty-eight hours later,
two sets of samples were stained with DAPI and analyzed
by fluorescence microscopy. Total RNA was isolated
from the other two sample sets using RNAqueous® MAG-96
and then converted to cDNA. Target cDNA levels were
analyzed in duplicate using a TaqMan Gene Expression
Assay for survivin (Hs00153353_m1). Data shown here
represent duplicate assays from single transfections.
Survivin mRNA levels were reduced 80% in the survivin
siRNA treated cells relative to control siRNA treated
cells. Knockdown of survivin induced morphological
changes to cell nuclei. The insets show representative
cells from the survivin and control siRNA treated samples.
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Summary
Thanks to optimized siRNA and real-time assay design using sophisticated bioinformatics
and design algorithms, performing gene silencing experiments is now easier
than ever. Pre-designed siRNA and qRT-PCR assay combinations are linked
through Ambion's website. The siRNA Database provides information about
Ambion's Silencer Pre-designed siRNAs targeting almost all human,
mouse, and rat genes in NCBI's RefSeq database. The siRNA database is also
a conduit to Silencer Validated siRNAs, which have been functionally
proven to reduce target mRNA levels by >70%. To find Silencer siRNAs
and TaqMan Gene Expression Assays to your gene of interest, visit www.ambion.com/siRNA.
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