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Representative Customer Results using SureSilencing™ RNA Interference shRNA Plasmids
Thousands of scientists have discovered the power of SureSilencing™ shRNA
plasmids. Published results from Satisfied SureSilencing™ shRNA plasmid
Customers are highlighted below.
Utilize the SureSilencing™ shRNA Plasmids today to
receive
- Guaranteed >70% Gene Knock Down
- Multiple shRNA plasmids to control for Off-Target Effects
- Renewable and Unlimited Source for Gene Knockdown
SureSilencing™ shRNA Plasmids - Applications
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Case Study - Stem Cell Research
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Case Study - Neurosciences Research
SureSilencing™ - Silencing Genes Beyond Humans
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Case Study - Mouse
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Case Study - Rat
Selection Screening Enhances Gene Knockdown
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Antibiotic Marker Selection Enhances Cell Profile
- Case Study - Antibiotic Marker Selection
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GFP Marker Serves Useful in Localization and FACS Sorting
- Case Study - GFP Marker Selection
Efficient Transfection of SureSilencing™ shRNA Plasmids
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SureFect/Liposomal Delivery
- Case Studies - Transfecting with SureFect Transfection Reagent
* Achieve Greater Transfection Efficiency
* Maintain Cellular Viability
* Apply to Variety of Cell Lines
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Electroporation
- Case Study - Transfecting with Electroporation
Conclusion
SureSilencing™ shRNA Applications in Various Fields
SureSilencing™ shRNAs have been used by researchers in a wide range of
research disciplines, including:
A. Stem cell research
B. Signal transduction
C. Neuroscience
D. Immunology
E. Cancer research
F. Reproduction
G. Infection
H. Hepatic disease
I. Gastrointestinal disease
J. Cardiac disease
K. Cell differentiation
Case Study - Stem Cell Research
Cells were treated with H2O2 (2 mM) with or without indomethacin (20 µM) for
24 hours. (A): Percentage of annexin V positive cells was analyzed by flow
cytometry. (B): Cleaved PARP was analyzed by Western blots. siRNA means the
siRNA generated by expression of shRNA plasmid.
Case Study - Neuroscience Research
Suppression of PUMA expression protects wild-type cortical neurons from
camptothecin-induced cell death. Wild-type cortical neurons were transfected at
day 1 after plating with shRNA plasmid which enables co-expression of the
respective shRNA and GFP. At 48 h after transfection, neurons were treated with
2.5 uM CPT or DMSO (vehicle control) and processed for GFP fluorescence and
nuclear staining (Hoechst 33258; blue) after 24 h. These results are
representative of two independent experiments.
SureSilencing™ - Silencing Genes Beyond Humans
The SureSilencing™ shRNA Plasmids specifically knock down the expression of
every human, mouse or rat gene by RNA interference.
Mouse - Case Study
Morphine Induces Defects in Early Response of Alveolar Macrophages to
Streptococcus pneumoniae by Modulating TLR9-NF-kB Signaling published in J.
Immunol. In their study, RNA interference for TLR9 Mouse cells were transfected
with either a negative control shRNA or SureSilencing™ shRNA plasmid for mouse
TLR9 according to our protocol. Also see above for Dr. Liou's stem cell study,
they used SureSilencing™ shRNA in mouse stem cells for their study
Also see:
Rat - Case Study
shRNA knockdown of HspB1 reveals a role in terminal differentiation and
filaggrin processing. A, Western analysis of highly differentiated (48 h post
confluent) REK cultures expressing a siRNA for HspB1 and a scrambled control.
The asterisk denotes the alteration in the expression of the mature filaggrin
subunit in the HspB1 siRNA expressing cell line. B, REK keratinocyte organotypic
cultures expressing the HspB1 siRNA exhibit a thicker cornified envelope.
Expression of loricrin is increased in the HspB1 siRNA culture. Keratin 10
expression is unchanged. The dotted line indicates the dermo-epidermal boundary.
Bar, 50 µm.
Successful Protocols from SureSilencing™ Customers
Successful inhibition of the target gene expression is essential for all
shRNA users. SureSilencing™ shRNA provide high knock-down efficiency for
target gene.
Antibiotic Marker Selection Enhances Cell Profile
With the choice of several antibiotic markers, generating stable cell lines
with multiple shRNA SureSilencing™ plasmids or introducing shRNA SureSilencing™
plasmids into existing stably transfected cell lines for further selection can
be performed with ease.
Case Study - Antibiotic Marker Selection
Knockdown of CAV1 expression with shRNA constructs prevents caveolae
formation and down-regulates the malignant phenotype of EWS cells in vitro. A,
immunoblot showing substantially reduced CAV1 levels in A4573 cells stably
expressing either of two shRNA constructs targeting different CAV1 sequences
(shCav1-1 and shCAv1-2), relative to mock-transfected or vector-transfected (shCont)
A4573 cells (lane 1). ?Actin was the loading control. B, electron micrographs
illustrating the virtual disappearance of caveolae from cells after
shRNA-mediated CAV1 knockdown (shCav1) compared with vector-transfected (shControl)
cells. C, phase-contrast micrographs taken from 50% to 60% confluent cultures of
the same cell types as in (B) illustrating the different morphologies and growth
patterns in culture. Inset, details of the different morphologies and growth
modalities. D, anchorage-independent growth assay showing data analysis from
triplicate cultures of parental A4573 (Mock), vector-transfected cells (shControl),
and cells stably expressing the two shRNA constructs indicated.
GFP Marker Serves Useful in Localization and FACS Sorting
The GFP marker included in the shRNA SureSilencing™ plasmids allows
researchers to conveniently estimate transfection efficiencies, track
transfected cells by fluorescence microscopy, and permits FACS-based enrichment
of transiently transfected cells.
Case Study - GFP
Effect of morphine on pneumococci-induced TLR9 dependent NFkB activation. B,
AMs were transfected with shRNA for TLR9, treated with either morphine (10 nM or
1 µM) or vehicle for 24 h, and infected with S. pneumoniae for 24 h. ELISAs
were performed on the cell supernatant to assess the protein levels of MIP-2. To
further confirm that morphine modulated TLR9-NF-kB signaling in AMs, MIP-2
production following S. pneumoniae infection was investigated using AMs
transfected with TLR9-GFP shRNA plasmid DNA. A high level of TLR9 expression was
seen in negative control shRNA-vector transfected AMs. Morphine treatment
decreased MIP-2 production following 4 h of infection. A significant lower level
of MIP-2 was shown in TLR9 shRNA plasmid DNA transfected AMs.
Transfection Reagents used for SureSilencing shRNA Plasmids
Transfection of SureSilencing™ shRNA plasmids into most common cell lines
can be done using different transfection reagents or transfection methods.
SureFect/Liposomal Delivery
As an advanced formulation, SureFECT™ transfection reagent can be used for
SureSilencing™ shRNA transfection in a variety of cell lines.
Case Studies - SureFect transfection reagent
SureFECT™ Reverse Transfects with Greater Efficiency than All Other
Traditional Transfection Reagents
In 96-well plates, 50 µL of diluted DNA (0.33 µg pCMVb-Gal plasmid) was
mixed with 50 µL dilutions of eight different commercial transfection reagents
in serum-free Opti-MEM. COS7 cells (15,000 in 50 µL) in normal medium
containing 5% FBS were then added to the wells for reverse transfection. Media
was changed 24 h post-transfection. Beta-gal enzymatic activity (OD570) was
assayed 32 h post-transfection utilizing 0.5 mg/ml CRPG as substrate.
SureFECT™ Maintains the Viability of Efficiently Reverse Transfected
Cells
In 96-well plates, 50 µL of diluted DNA (0.33 µg pCMVb-Gal
plasmid) was mixed with 50 µL dilutions containing four different amounts
of eight different commercial transfection reagents in serum-free Opti-MEM. COS7
cells (15,000 in 50 µL) in normal medium containing 5% FBS were then added to
the wells for reverse transfection. Media were changed 24 h post-transfection.
Viability was measured 32 h post-transfection utilizing an acidic phosphatase
assay.
SureFECT™ Works Equally Well On Multiple Cell Lines Commonly Used For
Gene Function Studies
SureFECT (0.3 µL per well) was used to reverse transfect MAPK1 siRNA (2
pmole) into different cell types in a 96-well plate. MAPK1 mRNA levels were
measured 48 h after transfection using quantitative real-time RT-PCR. The
knockdown efficiency (versus a negative control siRNA) is calculated via the
ΔΔCt method.
Electroporation
If your cells do not tolerate lipid-based transfection methods,
electroporation may be a useful alternative.
Case Study - Electroporation
The oxytocin-stimulated increase in intracellular calcium in PHM1 myometrial
cells was significantly suppressed by PLCB3 shRNA. (A) PHM1 cells were
electroporated with plasmids expressing no DNA, scrambled shRNA, or PLCB3sh1.
Fura-2 loaded cells were stimulated with oxytocin (100 nM) in the absence of
intracellular calcium. The data represent the mean ?SEM of data from all cells
selected for mGFP expression. The oxytocin response is reported as the peak
height of the initial calcium increase and integrated area of the calcium
transient, relative to scrambled control, for 185 (no DNA, gray bars), 131
(scrambled sequence, black bars) and 141 (PLCB3 shRNA, open bars) individual
cells collected in 3 separate experiments. (B) The effectiveness of PLCB3sh1 to
suppress PLCB3 mRNA was assessed in AD293 cells using a PLCB3 psiCHECK-2
reporter luminescence. The reporter (0.5 µg) and other plasmids (pUC19 plasmid
DNA, plasmid expressing scrambled shRNA sequence (scrambled), or PLCB3sh1, 1.5
µg) were transfected into AD293 cells as described in Methods.
Conclusion
SureSilencing™ shRNA provides a genome-wide RNAi tool for human, mouse, and
rat gene targets. It provides customers the flexibility for their studies with
different antibiotic selection markers. The high successful rate makes
customers' work easier and efficient. Our goal is to provide good tools for the
success of our customers in different research fields.
SureSilencing™ shRNA Plasmids specifically knock down the expression of
every human, mouse or rat gene by RNA interference. For each gene, we provide
four plasmids each with a different pre-designed short hairpin RNA (shRNA)
sequence. Our proprietary experimentally verified shRNA design algorithm insures
the maximum gene-specificity and efficacy. At least two of the four pre-designed
shRNA plasmids are guaranteed* to knock down expression of the targeted gene.
The availability of two effective sequences allows you to properly control for
non-specific and off-target effects.
Make SURE with SureSilencing™!
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