SiRNA Tools For Every Lab

siRNA Tools for Every Lab

• Silencer™ Kits for siRNA synthesis and labeling
• Transfection reagents
• Custom siRNA Synthesis Service
• Positive and Negative siRNA Controls

Twenty-one base double-stranded RNA molecules, termed small interfering RNAs or siRNAs, are potent mediators of gene silencing in mammalian cells. Early results from a number of laboratories suggest that with minimal effort, highly active and specific molecules for reducing expression of targeted mammalian genes can be developed. A successful siRNA research program requires a reliable source of high quality siRNA, and transfection agents optimized to deliver siRNA to mammalian cells at high efficiency. Ambion is pleased to introduce several new Silencer™ products for siRNA studies, including reagents for siRNA synthesis, labeling, and transfection; a custom siRNA Synthesis Service; and functionally tested Positive and Negative siRNA Controls. The complete line of Silencer siRNA products have been developed and supported by scientists at Ambion who are actively designing and performing silencing experiments of their own (Figure 1).

siRNAs at a Fraction of the Cost of Chemical Synthesis
In order to identify an siRNA that has a strong, gene-specific effect, it is often necessary to evaluate several siRNAs. Thus, a single gene silencing project can require 3 or more distinct siRNAs at a cost of $1500 or more when chemically synthesized. If you need to design siRNAs to more than one gene, the development costs can quickly reach tens of thousands of dollars. Now there is a more economical alternative. The Silencer siRNA Construction Kit (patent pending) synthesizes siRNA by in vitro transcription, producing transfection-ready siRNA at a fraction of the cost of chemical synthesis. For the price of one chemically synthesized siRNA, the Silencer siRNA Construction Kit can be used to produce 15 different siRNAs. Studies at Ambion have also demonstrated that siRNA produced with the Silencer siRNA Construction Kit is as much as 20X more potent than chemically synthesized siRNA of the same sequence. Thus each transcription reaction produces enough siRNA for hundreds of transfections (see "RNA Interference in Mammalian Cell Culture").  

Preparing an siRNA by transcription requires purchase of two inexpensive, desalted DNA oligonucleotides. As illustrated in Figure 2, these oligonucleotides include an 8 base sequence complementary to the 5' end of the T7 promoter primer included in the Silencer siRNA Construction Kit. (Ambion provides an online tool that automates the design of the required DNA oligonucleotides.) Each gene specific oligonucleotide is annealed to the T7 promoter primer. A fill-in reaction generates a double-stranded template ready for in vitro transcription. Complementary RNA products from the transcription reactions are hybridized, treated with DNase and RNase (to remove DNA template and polish the ends of the double-stranded RNA), and column purified. The entire procedure requires less than 24 hours (with less than 2 hours of hands-on time) and multiple siRNAs can be synthesized simultaneously. Each transcription/purification reaction produces enough siRNA for hundreds of transfections. The Silencer siRNA Construction Kit enables faster and more cost effective screening of potential targets within your gene so that the most potent siRNA is easily found.

The Silencer siRNA Construction Kit contains all reagents for transcription, hybridization, nuclease digestion and clean up (except your gene specific oligonucleotides for template construction). Also included is a detailed Instruction Manual, and control templates to produce both GAPDH and a scrambled siRNA. These controls can be used to verify that the kit is functional and can also be used for testing and troubleshooting transfection agents and protocols.

High Quality Custom siRNA Oligonucleotide Synthesis
Once you have identified specific siRNAs and optimized your siRNA protocols, it usually becomes economically more practical to have a few high purity siRNAs made by chemical synthesis. Typically a single synthesis will yield enough siRNA for the remainder of your research. Ambion is a licensed manufacturer of synthetic RNA oligonucleotides for siRNA research and will synthesize custom siRNAs at the 0.2 µM scale, which is enough siRNA for hundreds to thousands of transfections. As recommended in the literature, dTdT overhangs are added to the 3' ends of your siRNA, but you may choose another 3' sequence if you wish. To ensure that you receive a quality product, optimized deprotection and reverse phase desalting are provided at no charge. For the highest possible purity, a PAGE purification option is available that yields siRNA of greater than 90% purity. Various base modifications are also offered; please consult the Custom siRNA Synthesis page for more information.

In addition to delivering high quality synthetic siRNA, we have implemented several web-based tools to simplify siRNA design and ordering. Use Ambion's siRNA target design tool to choose a target sequence, then place your order using our web-based ordering form.

Effective Transfer of siRNA into Mammalian Cells
The efficiency with which different transfection agents deliver siRNA can vary dramatically both with the type of molecule being transfected (in this case, 21 bp double-stranded RNA) and the transfection agent employed. In fact, most commercially available transfection agents have been optimized to deliver DNA (plasmid or oligonucleotides) into cells and do not work well for siRNA. The few agents that are able to transfect RNA are optimized for mRNA transfection and exhibit disappointingly low efficiency when used with siRNA.

Ambion offers two transfection reagents to facilitate the transfer of siRNAs into mammalian cells, siPORT™ Amine (a polyamine mixture) and Lipid (a mixture of cationic and neutral lipids). These two agents have different transfection efficiencies for different cell types (summarized in Figure 3, below). Between these two transfection agents, a broad variety of cultured mammalian cells can be transfected with siRNAs.

The Silencer siRNA Transfection Kit contains both siPORT transfection agents and a well-characterized GAPDH synthetic siRNA, and is ideal for developing an optimal transfection protocol for your cells. The positive control GAPDH siRNA has been tested in human, mouse, rat, and monkey cell lines; a scrambled GAPDH Negative Control siRNA is also provided to confirm the specificity of the GAPDH siRNA and to demonstrate the absence of nonspecific toxic effect on your cells. The Silencer siRNA Transfection Kit contains sufficient reagents for over 200 transfections using 24 well tissue culture dishes. The siPORT Amine and siPORT Lipid Transfection Agents are also available separately in 0.4 ml and 1 ml sizes.

Proven siRNA Controls--Ready-to-Use
The gene specific Silencer siRNA Kits contain ready-to-transfect, chemically synthesized and gel-purified GAPDH, Cyclophilin, or c-myc siRNA. Enough siRNA is provided for as many as 150 transfections (of a single well in a 24 well plate). A corresponding scrambled siRNA negative control is also included. These kits are ideal for developing and optimizing your siRNA experiments and have been validated for use in mammalian cell lines.

For scientists who are already using the Silencer siRNA Construction Kit to produce their siRNA, Ambion recommends the Silencer GAPDH, Cyclophilin, or c-myc siRNA Control Templates. These templates, when used in conjunction with The Silencer siRNA Construction Kit, produce siRNA corresponding to Ambion's GAPDH, Cyclophilin, c-myc, and scrambled control sequences (discussed above). Ambion has also designed and tested three negative controls that have no significant homology to mouse, rat, or human gene sequences. These negative control siRNAs are ideal for controlling for non-specific effects on gene expression. Three different controls are offered in the unlikely event that one or another has an unanticipated effect on the expression pattern of your gene. Ambion's Negative Control siRNAs are available as ready to use chemically synthesized siRNA or as DNA templates for siRNA synthesis using the Silencer siRNA Construction Kit.

Versatile Cy3 and Fluorescein Labeling of siRNA:
The siRNA Labeling Kit
The Silencer siRNA Labeling Kits are ideal for labeling siRNA synthesized with the Silencer siRNA Construction Kit or with siRNA synthesized chemically. Labeled siRNA can be used to analyze the subcellular distribution of siRNA, in vivo stability, transfection efficiency, or expression attenuation. It can also be used with a labeled antibody in double label experiments to correlate siRNA uptake with down regulation of a target protein. The experiments discussed in "Visualizing siRNA in Mammalian Cells" use siRNA labeled with the Silencer siRNA Labeling Kit.

Two labeling kits are available -- for Cy3 or FAM (Fluorescein). Each Cy3 and FAM labeling kit comes with all the reagents necessary to label 65 µg of siRNA. The labeling reactions are scalable -- one (65 µg) to 12 siRNAs (5 µg each) can be labeled per kit. Research indicates that labeling siRNA does not affect its biological function.

Ambion is a licensed provider of siRNA, and the purchase of the Silencer siRNA Construction Kit confers permission to our customers for research use of siRNA.

Cy3 is a trademark of Amersham BioSciences.

The Silencer siRNA Labeling Kits contain reagents manufactured for Ambion by MIRUS.

siPORT Amine is manufactured for Ambion by MIRUS.