Create siRNA Expression Cassettes by PCR
Silencer™ Express

• Quickly test different siRNA sequence and promoter combinations before cloning into a vector

• Rapidly prepare siRNA expression cassettes by PCR -- no cloning, plasmid preps or sequencing necessary

• No need to synthesize siRNA in vitro

SECs are PCR products that consist of promoter and terminator sequences flanking a hairpin siRNA template. Upon transfection into cells, the hairpin siRNA is expressed from the PCR product and induces gene specific silencing. The Silencer Express Kits make it easy to produce siRNA expression cassettes (SECs) with a human H1, human U6, or mouse U6 promoter by PCR for use in RNAi studies.

Advantages of siRNA Expression Cassettes
In contrast to siRNA expression vectors, which require cloning and sequencing prior to use and thus take 1-2 weeks to prepare, SECs can be prepared in less than a day. SECs provide an excellent screening tool to find the most effective siRNA sequence, or to identify the best combination of promoter and siRNA in the experimental system being used (see Find the Best Promoter for Your Experimental System, at right). In fact, SECs provide the perfect complement to siRNA expression vectors. By incorporating restriction sites at their ends, SECs found to effectively elicit gene silencing can be readily cloned into a plasmid to create an siRNA expression vector with additional features such as an antibiotic selectable marker. The siRNA expression vector can then be used for stable siRNA expression in long term studies, or to enrich for cells that have taken up the vector -- an approach that is especially useful for hard to transfect cells.

Preparing SECs is Quick and Easy
Preparing SECs with the Silencer Express Kits is a simple, three-step process (Figure 1): (1) one or two oligonucleotides encoding the siRNA sequence are designed and ordered, (2) the oligonucleotide(s) are used as primers in one or more PCRs with the included RNA Pol III promoter-containing DNA template, and (3) the resulting PCR product is column-purified. The SECs are then transfected into cells.

 

Figure 1. Generating siRNA Expression Cassettes.

Everything You Need
for SEC Preparation and Purification
Ambion's Silencer Express Kits provide the reagents necessary to prepare 20 different SECs with either the human H1, human U6, or mouse U6 promoter. Each kit includes a double-stranded DNA template with one of the three promoters, PCR primers specific to the 5' and 3' termini of the SECs, and reagents to purify SECs for transfection. Both primers have modified ends to enhance SEC stability in mammalian cells. Also included are primers for generating SECs encoding an siRNA targeting GAPDH for use as a positive control and an siRNA with limited homology to the human or mouse genome for use as a negative control. For a complete solution, SuperTaq™ Polymerase is available separately.

 

Figure 2. Silencing c-fos with SECs Containing Three Different Polymerase III Promoters. siRNA Expression Cassettes containing the mouse U6 (Mo-U6), human U6 (Hu-U6), and human H1 (Hu-H1) promoters and encoding a c-fos-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the cells were assessed using nuclear staining with DAPI and immunofluorescence using a c-FOS antibody. Non-transfected cells (NT) as well as cells transfected with an SEC expressing a negative control siRNA (scrambled) demonstrate wild-type levels of c-FOS. The relative level of reduction in gene expression was quantified and is provided in the bar graph.

SuperTaq and SuperTaq Plus are trademarks of and are manufactured by Enzyme Technologies Ltd. and sold under licensing arrangments with F. Hoffman­La Roche, Ltd., Roche Molecular Systems, Inc. and the Perkin­Elmer Corporation. Ambion, Inc. is a distributer of Enzyme Technologies, Ltd., except in the following countries: Austria, Benelux, Denmark, Greece, Sweeden, Switzerland, Taiwan, and the United Kingdom.

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