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From: Ambion Inc - The RNA Company []
Sent: Monday, April 28, 2003 2:52 PM
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Gene Silencing
with PCR Products

Despite the enormous potential of siRNA expression vectors for gene silencing experiments, they require time consuming and laborious cloning and purification prior to use, often taking 1-2 weeks to prepare. Recently, Castanotto and colleagues reported the use of PCR products to express siRNA hairpins within cells. Ambion has developed three kits, each with different RNA polymerase III promoters, based on this technology. The new Silencer Express siRNA Expression Cassette Kits make it easy to produce siRNA expression cassettes (SECs) by PCR in less than a day. These SECs can be introduced into cells to induce gene silencing — without prior cloning or sequencing.

SECs prepared by the Silencer Express Kits provide an excellent screening tool to find the most effective siRNA sequence. Because three different promoters — human H1, human U6 and mouse U6 — are available, the kits are ideal for quickly identifying the best combination of promoter and siRNA sequence in your experimental system. In fact, SECs provide the perfect complement to siRNA expression vectors. SECs found to effectively elicit gene silencing can be readily cloned into a plasmid to create an siRNA expression vector for long term studies.

To learn more about this exciting technology,
click here.
Five Ways to Produce siRNAs —
Choose the Best Method for Your Research
Currently, there are five methods for generating siRNAs for gene silencing studies:

1. Chemical synthesis

2. In vitro transcription

3. Digestion of long dsRNA by an RNase III family enzyme

4. Expression in cells from an siRNA expression plasmid or viral vector

5. Expression in cells from a PCR-derived siRNA expression cassette

For researchers just beginning to perform gene silencing studies, having to choose among the methods can be downright intimidating. Certainly each one has its advantages and disadvantages. Methods 1–3 involve in vitro preparation of siRNA that is then introduced directly into mammalian cells or animals by transfection, electroporation, injection, or other method. Methods 4 and 5 rely on the introduction of DNA-based vectors and cassettes that express siRNAs within the cells. Which method is best will depend on the goals of the experiment and the experimental system (e.g., cultured cells versus animals). Ambion has written a new article that briefly describes the five methods, presents their advantages and disadvantages, and discusses the types of applications for which they are best suited. Read it here.
RNAi 2003 Meeting in Boston
May 5-6, 2003
Dr. David Brown, Ambion's Associate Director of Research and leader of one of Ambion's research teams working on RNAi related technologies, will be speaking at the RNAi 2003 Boston Meeting on May 5th. This meeting, officially titled "RNA Interference Technology in Drug Validation and Development", includes RNAi related lectures by respected scientists from both academia and industry. Whether you are just starting out with gene silencing experiments, or you're an RNAi expert, you're bound to learn something new at this two day meeting.
For official meeting information, please click here.

Dr. Brown recently participated in a panel discussion, "Gene Silencing by RNAi" on The discussion took place on March 23rd, and also included Drs. Dmitry Samarsky (Sequitur) and Natasha Caplan (NHGRI). Topics included the future of RNAi research and its potential for animal studies. Wonder what the panelists said? Satisfy your curiosity. Transcripts and a streaming audio recording of the discussion are available here (free registration required for access).
Measure Gene Silencing Effects
by RT-PCR Without RNA Isolation
There are several different ways to measure gene silencing effects. For instance, RNA levels can be monitored by Northern blot, array analysis, or RT-PCR; protein levels can be assessed by immunofluorescence or Western blot; and finally phenotypic effects can be monitored by enzymatic assay or even by direct observation. One of the most sensitive ways to measure changes in target gene expression is by real-time RT-PCR. This assay has an added advantage in that it can be readily adapted for high throughput. But RT-PCR, like the other RNA analysis procedures, has traditionally required RNA isolation prior to assay. Ambion's Cells-to-cDNA™ II Kit avoids RNA isolation altogether, permitting the analysis of gene expression levels in cell lysates by real-time RT-PCR. Ambion scientists have used this kit to analyze silencing of cdc-2, GAPDH and survivin in HeLa cells after transfection of siRNA. Read about these experiments and see the data here.
RNAi Resources
Ambion has made it easy to keep up with the rapidly advancing RNAi field. The RNA Interference Resource lists the most relevant new scientific articles, the latest information from our own labs, links to must-see web sites, and details on the newest tools and protocols to help you in your RNAi research. Visit often because we update the resource frequently.
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©Copyright 2003 Ambion, Inc.



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