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The SMARTchoice platform extends of the classic SMARTvector 2.0 design to incorporate the most ideal promoter for your specific cells of interest. Because efficient gene silencing depends on both the design and level of expression of the shRNA, it is critical to choose silencing reagents where both the targeting sequence and the specific promoter driving expression are taken into consideration.
The SMARTchoice shRNA platform saves time and money by allowing them to make informed decisions. First, evaluate activity of multiple promoters in the target cells of interest. Next, order SMARTvector 2.0 gene-specific lentiviral shRNA and controls containing the desired promoter and fluorescent reporter.
Learn more about SMARTvector 2.0 design
Promoter activity varies between cell types
Promoter activity correlates to silencing potential
Thermo Scientific SMARTchoice shRNA platform extends the advantages of innovative SMARTvector 2.0 design for enhanced functionality with greater flexibility.
Begin by selecting the ideal promoter for your RNAi experiment using the SMARTchoice shRNA Promoter Selection Plate, then order gene-specific and control shRNAs as concentrated, transduction-ready lentiviral particles customized for your cells of interest.
The recommended workflow for carrying out RNAi experiments using the SMARTchoice shRNA platform is illustrated below in Figure 1.
Figure 1. Recommended workflow for SMARTchoice shRNA experiments. Identify the optimal promoter for your cells of interest with the transduction-ready SMARTchoice Promoter Selection Plate. Next, place an online order for SMARTvector 2.0 shRNA constructs containing your choice of promoter and fluorescent reporter and save the extensive time, labor and money required for production of high-titer lentiviral particles. Benefit from our internal, quality-controlled process of cloning and packaging your customized vector into lentiviral particles using the Thermo Scientific Trans-Lentiviral Packaging System for enhanced biosafety.
Plan practical RNAi experiments using the optimal promoter choice for your specific cells. In a single matrixed experiment, the SMARTchoice Promoter Selection Plate enables the evaluation of seven different promoters at a range of multiplicities of infection (MOIs) in your cells of interest.
ORDER SMARTchoice shRNA Promoter Selection Plate
Promoter activity cannot necessarily be predicted based on species. For example, our scientists observed a mouse-derived promoter to be more active than a human-derived promoter in some human cell lines (see Figure 4.).
The 96-well SMARTchoice shRNA Promoter Selection Plate Layout
The SMARTchoice shRNA Promoter Selection Plate enables straightforward qualitative assessment of promoters that actively drive expression
Selection of mouse CMV promoter for greatest gene silencing
Cross-function of mouse promoter in human A549 cells
SMARTchoice shRNA Controls are SMARTvector 2.0 positive and negative controls which include your choice amongst seven different promoters and two fluorescent reporters. If the optimal promoter for your specific cells of interest has yet to be identified, we recommend utilizing the SMARTchoice shRNA Promoter Selection Plate to evaluate multiple promoters in a single experiment.
Consistent Results Across Cell Lines
Figure 1. Promoter activity varies across several human and rodent cell lines. Cells were plated at a density of 50,000 cells per well in a 24-well plate and transduced at MOI = 15 with SMARTvector 2.0 Empty Vector Control Particles expressing TurboGFP. Promoter activity was assessed at 72 hours post-transduction by the fluorescence intensity of TurboGFP.
Figure 2. Promoters driving the expression of TurboGFP have differential activity from cell to cell. Multiple cell lines were transduced with SMARTvector Non-targeting shRNA Control Particles which delivered vectors expressing TurboGFP driven by seven different cellular and viral promoters. Control wells contained untreated cells. Fluorescent images were obtained 72 hours post-transduction. Visual assessment of TurboGFP expression in human PC-3, human OVCAR-8, human Jurkat and mouse NIH/3T3 cells shows a varying degree of promoter activity across cell lines.
Figure 1. The 96-well SMARTchoice shRNA Promoter Selection Plate layout.
Figure 2. Human A549, HEK293T and Jurkat cells were transduced with concentrated lentiviral particles arrayed in the SMARTchoice Promoter Selection Plate. TurboGFP expression was assessed by fluorescence microscopy 72 hours post-transduction. Images clearly demonstrate that the most functional promoter in A549 cells is mCMV, whereas the hCMV promoter is most active in HEK293T, and the mEF1α promoter is most active in Jurkat cells. TU = transducing unit.
Figure 3. Selection of the mouse CMV promoter for mouse NIH/3T3 cells using the SMARTchoice Promoter Selection Plate results in greater gene silencing. Gene silencing experiments targeting mouse GAPDH and RHOA genes demonstrate that shRNAs expressed by the mCMV promoter are significantly more effective at silencing the target gene in NIH/3T3 cells when compared to the same shRNAs expressed by the hCMV promoter. Cells were transduced with lentiviral particles at MOI = 20, 10 and 5; gene expression levels were detected and normalized to controls 72 hours post-transduction using Thermo Scientific Solaris qPCR Gene Expression Assays and Master Mix.
Figure 4. shRNAs expressed from the mouse CMV promoter result in significant gene silencing in human A549 cells. One shRNA targeting human GAPDH and three shRNAs targeting human RHOA were delivered individually to A549 cells with expression driven by either the mCMV or hCMV promoter. Results demonstrate that all four shRNAs expressed from the mCMV promoter significantly silenced GAPDH or RHOA compared to the same shRNAs driven by the hCMV promoter. Cells were transduced with lentiviral particles at MOI = 20, 10 and 5; gene silencing was measured using Solaris™ qPCR Gene Expression reagents 72 hours post-transduction.
Figure 1.SMARTvector 2.0 Lentiviral shRNA Controls: Non-Targeting Control and GAPD. K562, HUVEC and SH-SY5Y cells were transduced at three MOIs and assessed for mRNA knockdown 72 hours post-transduction using a branched DNA assay (Panomics, Inc.)