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amaxa eNews #3
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Nucleofection: The Method of Choice for all RNAi Applications
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Gene silencing by RNA interference (RNAi) is a powerful technology for assessing gene function in mammalian cells and has greatly accelerated basic research, functional genomics, and target discovery and validation. Various RNA and DNA substrates can be used in RNAi experiments: siRNA oligonucleotides, shRNA vectors, and miRNA substrates. Clearly, in addition to the ability of the siRNA sequence to specifically silence the target mRNA, the efficiency with which the RNA or DNA vector can be transfected into the cells of interest is crucial to the success of any RNAi experiment. With up to 99% efficiency for siRNA delivery and up to 90% efficiency for DNA delivery, nucleofection is the delivery method of choice for any RNAi substrate and any cell type.
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Nucleofection and RNAi – Benefits at a Glance
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| Efficient non-viral delivery | RNAi experiments in difficult-to-transfect cell types, e.g., Jurkats or primary neurons
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| | High transfection efficiencies for different substrates, e.g., RNA oligonucleotides (siRNA, synthetic miRNA, miRNA inhibitors) and shRNA- or miRNA-expressing vectors
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| Optimized Protocols | Ready-to-use protocols for primary cells and cell lines
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| Same protocol for different substrates | Easy switch of substrates
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| | Co-transfection of siRNA oligonucleotides with plasmid DNA for transfection control or rescue experiments
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| 96-well Shuttle | High-throughput RNAi screenings in primary cells and difficult-to-transfect cell lines
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| Non reagent-based transfection | No lipid-induced off-target effects [1]
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| | No lipid-induced induction of interferon responses [2]
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| | High reproducibility
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Thanks to the Nucleofector 96-well Shuttle System, high-throughput RNAi library screenings using siRNA or shRNA can now be performed using virtually any cell line as well as primary cells. For the first time, target identification and validation can be performed using primary cells, drastically increasing result significance. High transfection efficiencies combined with high cell viability and low well-to-well variances make the Nucleofector 96-well Shuttle System the ideal tool for siRNA and shRNA library screening.
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Figure 1: Nucleofection outperforms lipofection for effective GAPDH mRNA knockdown in hard-to-transfect cell types.
Cells were transfected with 5 pmol SMARTpool reagent targeting GAPDH using the 96-well Shuttle (according to the respective optimized protocol) or Reagent L (after titration of optimal reagent amount). Negative control samples were transfected with 5 pmol siCONTROL Non-targeting siRNA #1. Twenty-four hours post transfection, cells were analyzed for mRNA expression by Quantigene branched DNA assay (Panomics). GAPDH mRNA levels were normalized to cyclophilin B and to siCONTROL samples. (Data generated in collaboration with Thermo Fisher Scientific, Dharmacon Products).
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High transfection efficiencies and knockdown using siRNA oligonucleotides
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More than 300 publications in top ranking journals have reported successful target gene knockdown using nucleofection for delivery of siRNA oligonucleotides. High transfection efficiencies of up to 99% have been observed (Figure 2). Depending on the cell type and target, efficient knockdown can be observed at siRNA concentrations lower than 10 nM (Figure 3). Furthermore, the siRNA amount has no influence on cell viability (Figure 3A).
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Figure 2: Up to 99% delivery of siRNA duplexes by nucleofection.
Different suspension cell lines were transfected using the appropriate Nucleofector Kit and rhodamine-labeled siRNA duplexes. 3 hours after delivery, cells were analyzed by light and fluorescence microscopy.
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Figure 3: Efficient mRNA down-regulation with less than 1 pmol siRNA in suspension cell lines and primary cells.
Using the 96-well Shuttle, Jurkat clone E6-1 (ATCC TIB-152) (A) or HUVECs (B) were nucleofected with various amounts of SMARTpool siRNA reagents (Dharmacon) targeting GAPDH (A) or vimentin (B). The control sample is a Dharmacon siCONTROL Non-Targeting siRNA Pool, while “untreated” samples are cells that received neither siRNA nor nucleofection. mRNA levels were analyzed 24 hours post transfection by the QuantiGene branched-DNA assay (Panomics) and normalized to siCONTROL sample. Cell viability (only shown in A) was determined 48 hours post-transfection by the CellTiter-Blue assay (Promega) and normalized to untreated cells. (Data generated in collaboration with Thermo Fisher Scientific, Dharmacon Products).
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For guidelines and further information about critical parameters for successful siRNA nucleofection please refer to our Application and Tech Notes.
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Efficient delivery and mRNA knockdown using shRNA vectors
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In addition to or as an alternative to siRNA duplexes, shRNA vectors are frequently used for RNAi applications. These vectors offer an advantage over siRNA duplexes in that they permit a longer transient knockdown of the target mRNA and can be stably integrated into the target cells' DNA.
Nucleofection achieves up to 90% transfection efficiency of DNA vectors into cell lines and primary cells. The technology therefore significantly increases the range of cell types in which plasmid-based RNAi systems can be used, including primary neurons and Jurkats. This makes it an ideal alternative to circumvent laborious viral transduction methods. Many experiments have shown successful gene silencing following nucleofection of shRNA vectors (Figures 4 and 5).
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Figure 4: Gene silencing in primary neurons by transfection of an shRNA vector — Quantitive down-regulation of CDC10 protein.
Rat hippocampal neurons (E17) were transfected with the shRNA vector pSuperior targeting CDC10 using the 96-well Shuttle. A and B: Efficient nucleofection of pSuperior is shown by eGFP expression after 1 day in vitro. C: Immunostaining of CDC10 (red fluorescence) shows reduced endogenous CDC10 protein levels in transfected neurons (green) after 4 days in vitro compared to untransfected cells (red). Western blot analysis (D) and quantification (E) of CDC10 downregulation. (Data courtesy of Prof. Kiebler, Medical University of Vienna, Vienna, Austria.)
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Figure 5: Efficient knockdown using shRNA vectors.
PAM212 cells were nucleofected with Fat1 RNAi plasmid and doubly immunostained for Fat1 (red) and (A) beta-catenin or (B) F-actin (green) 2 days after transfection. More than 95% of the cells were nucleofected and showed a significant reduction in the level of Fat1 protein. Nucleofected areas lacking Fat1 show looser cell-cell associations (A) and a disrupted actin organisation (B, arrows; arrowheads: cell junctions in Fat1-positive cells). (Tanoue et al., reproduced from The Journal of Cell Biology, 2004, 165(4), 517 by copyright permission of The Rockefeller University Press and by permission of the authors.)
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Nucleofection of miRNA substrates into suspension cell lines and primary cells
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MicroRNAs (miRNAs) – endogenous, small, non-coding RNA molecules – are key regulators of gene expression at the level of translation. They are differentially expressed in tissues, critical in the development of organisms, involved in viral infection, and associated with oncogenesis. Their functionality can be analyzed by overexpression using transfection of synthetic miRNAs or miRNA-expressing plasmids or by down-regulation using transfection of miRNA inhibitors. Nucleofector Technology allows this class of genetic elements to be studied in difficult-to-transfect cell lines and primary cells (Figure 6).
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Co-transfect with ease
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Some RNAi approaches require efficient co-transfection of siRNA oligonucleotides together with DNA vectors, e.g., rescue experiments or labeling of transfected cells (transfection control). Nucleofection is ideal for such applications as it is substrate independent, allowing siRNA and DNA vectors to be transfected using the same protocol (Figure 6, Figure 7). It therefore overcomes the limitations of reagent-based methods which require differing conditions for different substrate types. One example is the siRNA Test Kit which allows fast establishment of siRNA experiments. It is based on co-transfection of pmaxGFP and an siRNA duplex directed against maxGFP (Figure 7). The siRNA test Kit gives you a first idea whether the nucleofection itself as well as the RNAi pathway in general works in your cell-type of interest.
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Figure 6: Co-transfection of neurons, maintenance of functionality — Expression of miR132 induces neurite sprouting by targeting a protein that represses neurite outgrowth (p250GAP).
Rat neonatal cortical neurons were transfected with a GFP reporter (green) and co-transfected with vector control, or expression constructs for premiR1-1 premiR132 using Rat Neuron Nucleofector Kit and Nucleofector Device. Cells were immunostained for the neuronal marker MAP2 (red). Only cells transfected with premiR132 show neurite sprouting. (Vo et al., reproduced from the Proc Natl Acad Sci USA, 102(45): 16429 by copyright of the National Academy of Science and by permission of the authors.)
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Figure 7: Example of nucleofection of NIH/3T3 cells with pmaxGFP and siRNA.
NIH/3T3 cells (ATCC CRL-1658) were nucleofected with 0.5, 1 or 2 μg of pmaxGFP only (A, B, C top row) or co-transfected with 0.5, 1 or 2 μg pmaxGFP and 1.5 μg siRNA directed against maxGFP (D, E, F bottom row). Gene silencing of maxGFP expression was monitored by fluorescence microscopy, 24 hours post nucleofection.
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Ordering information
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| Cat.No. | Devices | | AAD-1001 | Nucleofector II Device | | AAM-1001 | 96-well Shuttle | | | | | | Nucleofector Kits for Primary Cells* | | | | | | Related Products | | VSC-1001 | siRNA Test Kit² | | | | | | Nucleofector Kits for Cell Lines | | VCA-1001 | Cell Line Nucleofector Kit R | | VCA-1002 | Cell Line Nucleofector Kit T | | VCA-1003 | Cell Line Nucleofector Kit V | | VCA-1004 | Cell Line Nucleofector Kit C | | VCA-1005 | Cell Line Nucleofector Kit L | | VCO-1001 | Cell Line Optimization Nucleofector Kit | | | | | | 96-well Nucleofector Kits for Primary Cells* | | | | | | 96-well Nucleofector Kits for Cell Lines | | VHCA-1001/ VHCA-2001 | Cell Line 96-well Nucleofector Kit SE (96 reactions/960 reactions) | | VHCA-1002/ VHCA-2002 | Cell Line 96-well Nucleofector Kit SF (96 reactions/960 reactions) | | VHCA-1003/ VHCA-2003 | Cell Line 96-well Nucleofector Kit SG (96 reactions/960 reactions) | | VHCO-1001 | Cell Line Optimization 96-well Nucleofector Kit |
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*Click here for an up-to-date list of all Nucleofector and 96-well Nucleofector Kits for Primary Cells.
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References
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[1] Fedorov Y et al (2005) Nat Methods 2(4): 241
[2] Sioud M (200) JMolBiol 348(5): 1079-90
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The Nucleofector Technology, comprising Nucleofection Process, Nucleofector Device, Nucleofector Solutions, Nucleofector 96-well Shuttle System and 96-well Nucleocuvette plates and modules is covered by patent and/or patent pending rights owned by amaxa AG. amaxa, Nucleofector, nucleofection, maxGFP, 96-well Shuttle and Nucleocuvette are either registered trademarks or trademarks of amaxa AG in the U.S. and/or Germany and/or other countries.
Dharmacon, SMARTpool and siCONTROL are registered trademarks of Dharmacon, Inc. ATCC and the ATCC Catalog Marks are trademarks of ATCC used under License. Apo-ONE and CellTiter-Blue are registered trademarks or trademarks of Promega Corporation in the U.S. and/or other countries. BLAST is a registered trademark of the National Library of Medicine. Other product and company names mentioned herein are the trademarks of their respective owners.
²siRNA technology licensed to QIAGEN is covered by various patent applications, owned by the Massachusetts Institute of Technology, Cambridge, MA, USA and others. This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to be used as positive control with this amaxa products only. Any use of proprietary nucleic acid or fluorescent protein other than as positive control with this amaxa product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at license@evrogen.com.
amaxa disclaims all warranties, whether expressed or implied, including any warranty as to the quality, accuracy, safety, or suitability of the information provided in this e-newsletter for any particular purpose. Any use of the information contained on any page of this e-newsletter is evidence of agreement with these terms of use.
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