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siRNA-Mediated Gene Silencing
in Mammalian Cells
Richard A. Jarvis, Vince Pallotta,
Mike Byrom, David Brown, and Lance P. Ford
Ambion, Inc., 2130 Woodward Street, Austin, TX 78744-1832
Highlights
- Over half of the siRNAs tested provide
greater than 50% reduction in target gene product
- Positive correlation exists between mRNA
and protein suppression in genes targeted by siRNAs
- Enzymatically prepared siRNAs proved to
be more potent than chemically synthesized siRNAs
- siRNA-induced gene silencing occurs in
diverse mammalian cell types
Introduction
Multiple small interfering RNAs (siRNAs) complementary
to c-myc and GAPDH mRNA were evaluated to establish parameters
for siRNA design, to optimize transfection procedures for short,
double-stranded RNAs, and to develop a new method for siRNA synthesis.
Using a novel method of siRNA synthesis, four siRNAs were prepared
that spanned the length of each target gene. These siRNAs were
transfected into several types of mammalian cells and the relative
reduction in target RNA and protein was determined. siRNAs to different
target sites provided different levels of mRNA and protein knockdown.
Spatial orientation along the length of the mRNA appeared not to
be a factor in dictating siRNA activity, but target sites with
lower GC content tended to be more susceptible. Less in vitro transcribed
siRNA than chemically synthesized siRNA was needed for transfection.
The most effective GAPDH siRNA was used to develop transfection
protocols for various cell types. The novel method of siRNA synthesis
used here provides a cost effective alternative to chemical synthesis.
Experimental Method
- siRNAs to human GAPDH, c-myc, and La were designed following
the procedure described by Elbashir et al. (2001).
- siRNAs were prepared either by in vitro transcription using
the Ambion Silencer™ siRNA
Construction Kit or by chemical
synthesis.
- After the addition of siRNAs and transfection reagent, cells
were incubated 8-72 hr before harvesting and analysis.
- Target mRNA expression levels were determined by either Northern
analysis using the NorthernMax-Gly
Kit (Ambion) or by real-time RT-PCR on the ABI7700 with dual
labeled probe detection.
- Protein levels were measured in experimental samples by Western
analysis or by immunofluorescence analysis (antibody
details).
- Cell proliferation assays were performed using Alamar Blue
(Biosource International, Inc.).
Results
- Seven out of 12 siRNAs reduced the expression of the target
gene by at least 50% and by as much as 94% (Table 1).
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| Table 1. Reduction
in Target mRNA and Protein Induced by siRNAs. |
- siRNAs to different sequences within a gene are not equally
effective at reducing gene expression. (Figures 1 and 2).
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| Figure 1. Locations
of the siRNAs in c-myc mRNA. The
c-myc mRNA is depicted as a line that is divided into 5'
UTR, coding region, and 3' UTR. Also shown are the start
and stop codons and the location of the phosphorothioate
oligonucleotide. |
| A |
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| B |
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| Figure 2. siRNAs
have Differential Abilities to Induce RNAi. (A)
siRNAs designed to different regions of the c-myc RNA sequence
were transfected into the HeLa S3 cells and analyzed 48
hr after transfection for changes in proliferation and
protein levels. Cell proliferation rates were analyzed
using the Alamar Blue assay. (B) Immunofluorescence analysis
of c-myc protein in HeLa S3 cells 48 hr following transfection
with buffer only or with the siRNA against the 3' UTR. |
- The extent of gene silencing is apparent at both mRNA and protein
levels. (Figures 2, 3 and 4).
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| Figure 3. Reduction
of GAPDH protein by in vitro transcribed siRNA. Immunhistochemistry
staining of BJ fibroblast cells with fluorescein-labeled
monoclonal antibody to GAPDH and DAPI nuclei staining 48
hr following transfection with scrambled control siRNA
or siRNA against GAPDH. |
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| Figure 4. Susceptibility
of Various Mammalian Cells to siRNA-induced Gene Silencing. The
indicated cell types were transfected with a chemically
synthesized (Panel A) or in vitro transcribed (Panel B)
siRNA specific to GAPDH. The cells were harvested 48 hr
after transfection. RNA was isolated, fractionated by denaturing
gel electrophoresis, and transferred to a nylon membrane.
The Northern blots were hybridized with RNA probes specific
to GAPDH, cyclophilin, and 28S rRNA. The 28S rRNA probe
was made to significantly lower specific activity. |
- siRNAs suppressed intended targets with no apparent nonspecific
effects on unrelated genes. (Figure 4).
- All cell types tested were susceptible to some level of silencing
by the GAPDH siRNA. (Figure 4).
- The optimal concentration for transfection of in vitro transcribed
siRNA was consistently at least 10-fold lower than that for chemically
synthesized RNA. (Figure 5).
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| Figure 5. Potency
of siRNAs Prepared by Chemical or Enzymatic Synthesis. HeLa
cells were transfected with the indicated concentration
of GAPDH siRNA prepared by chemical synthesis (synthetic)
or in vitro transcription. Both siRNAs were made to the
same sequence of the mRNA. GAPDH mRNA levels from the various
samples were assessed by Northern analysis. GAPDH signals
were qualified using a phosphorimager and normalized using
signal from a cyclophilin probe. |
Discussion
siRNA-induced gene silencing in
mammalian cells shows great promise as a tool to regulate the
expression of specific genes. In the current study, 4 siRNAs
to each of 3 target genes were prepared and tested. Strikingly,
over half of the siRNAs reduced the expression of the desired
gene by at least 50%. Furthermore, at least 1 of the 4 siRNAs
tested for each target significantly decreased the expression
of the targeted gene. Further work may help determine why some
sequences are not active, ultimately leading to more reliable
design criteria. Importantly, RNA and protein analysis suggests
that only the targeted gene is affected by the corresponding
siRNA. Taken together, these results indicate that with minimal
effort, highly active and specific siRNAs for reducing targeted
mammalian gene expression can be developed.
Reference
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl
T (2001) Nature 411: 494-498.
Antibodies
- The mouse monoclonal antibody to GAPDH was from RDI (Cat #RDI-TRK5G4-6C5).
- The mouse monoclonal antibody to La was from BD Transduction
Laboratories (Cat #610904).
- The mouse monoclonal antibody for c-myc was from NeoMarkers
(c-myc Ab-5 clone 67P05).
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