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RNAi: A How To for New Users

mammalian cultured cells, RNAi is typically induced by siRNA introduced directly or expressed as a hairpin structure from a DNA construct within the cells. Currently, there are six methods for generating siRNAs:

In vitro preparation of siRNA

1. Chemical synthesis
2. In vitro transcription
3. Digestion of long dsRNA in vitro by RNase III or Dicer

Introduction of DNA-based vectors and cassettes that express siRNAs within cells

4. Expression from a plasmid
5. Expression from a viral vector
6. Expression from a PCR product

All of these methods, except creation of siRNA populations by digestion of long dsRNA, require careful design of the siRNA to maximize silencing of the target gene while minimizing the effects on off-target genes. Ambion supports all six siRNA synthesis methods with high quality kits and reagents. See the article in a previous issue for more details about each of these techniques and to determine the one best suited to your experimental needs.


siRNAs for Transient Transfection: Chemical siRNA Synthesis
Currently, the most widespread application of RNAi involves transient transfection of cultured mammalian cells followed by a downstream assay to monitor the RNAi effect. For this application, chemical synthesis is th
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