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Sigma-Aldrich Research Biotechnology
Microinjection of siRNA Protocol
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Sigma® has prepared product lists for commonly used protocols from Cold Spring Harbor Protocols. |
View the entire protocol on BioSupplyNet.com.
Selected Protocol from "Gene Transfer: Delivery and Expression of DNA and RNA",
Edited by Theodore Friedmann and John Rossi. |
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| Genetic Manipulation of Mammalian Cells by Microinjection |
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David W. Rose
Department of Medicine and Moores Cancer Center, University of California,
San Diego, La Jolla, California 92093-0673 |
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ABSTRACT
Of the many methods available for the introduction of DNA and other biological materials into cells, microinjection of individual mammalian cells is one of the more technically difficult to perform. The technique is also reliant upon comparatively expensive equipment and demands the experimental skills of an experienced operator. There are, however, very good reasons to use this approach. It allows the introduction of molecules into a defined population of cells at a defined concentration, and the timing of the experiment can be controlled stringently, minimizing problems associated with overexpression. Perhaps the most powerful aspect of microinjection is the ability to introduce several types of reagents into cells simultaneously, including DNA constructs, a labeled dextran to mark the injected cells, antibodies, short interfering RNAs (siRNAs), and peptides (Lavinsky et al. 1998; Rose et al. 1998; Jepsen et al. 2000; Zhu et al. 2006). No other techniques available provide these capabilities.
Delivery by microinjection can be used for any type of cell that is adherent in culture, including primary cells. Because siRNA can be easily and rapidly generated for any target gene, it is relatively simple to assess many effects of the knockout of any gene in any type of adherent cell in a matter of a few days. In addition, delivery by microinjection assures that every cell receives the siRNA at a relatively equal concentration. Many of the disadvantages of the approach relate to the challenging aspects of the technique itself: It is not trivial to learn, and it is an undertaking that requires attention to detail. It also suffers the technical disadvantage that a limited number of cells are involved, which often does not permit the subsequent analysis of effects upon some biochemical parameters. The major limitation of the approach is the small amount of material obtained, but as the sensitivity of analysis techniques increases, so does the usefulness of this method. |
| Products Available for this Protocol |
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Buffers, Solutions, and Reagents |
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| Dextran, fluorescently labeled |
46945 |
Fluorescein isothiocyanate–dextran |
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| Oligonucleotide siRNAs |
siRNAs |
Oligonucleotide siRNAs |
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| 5 mM Sodium phosphate |
342483 |
Sodium phosphate |
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| KCl |
P9541 |
Potassium chloride |
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| Glucose |
G5400 |
D-(+)-Glucose |
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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
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