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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

  • Case Study - Stem Cell Research
  • Case Study - Neurosciences Research

SureSilencing™ - Silencing Genes Beyond Humans

  • Case Study - Mouse
  • Case Study - Rat

Selection Screening Enhances Gene Knockdown

  • Antibiotic Marker Selection Enhances Cell Profile
       - Case Study - Antibiotic Marker Selection
  • GFP Marker Serves Useful in Localization and FACS Sorting
       - Case Study - GFP Marker Selection

Efficient Transfection of SureSilencing™ shRNA Plasmids

  • SureFect/Liposomal Delivery
    - Case Studies - Transfecting with SureFect Transfection Reagent
       *  Achieve Greater Transfection Efficiency
       *  Maintain Cellular Viability
       *  Apply to Variety of Cell Lines
  • 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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