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Silencer Validated siRNAs

Silencer Validated siRNAs are single siRNA duplexes that have been verified by real-time RT-PCR to reduce the expression of a number of important human genes. The cut-off for siRNA validation is a >70% reduction in target gene expression 48 hours post-transfection, though the average reduction in target gene expression in the inital set of validated siRNAs is greater than 90%. Every Silencer Validated siRNA strand is purified by HPLC, tested for purity by capillary electrophoresis or analytical HPLC, and verified for identity by MALDI-TOF mass spectometry. After annealing, the siRNAs are analyzed by nondenaturing gel or capillary electrophoresis to confirm that the strands are properly annealed. The result is the highest quality siRNA with a sequence verified to reduce gene expression.

Cenix and the Genome-Wide siRNA Library
The validated siRNAs described here are the first of what will be many new products stemming from Ambion's collaboration with Cenix BioScience. Cenix was founded in 1999 and pioneered the use of genome-wide dsRNA libraries by targeting all of the genes in the C. elegans and Drosophila genomes (1). Cenix was the first company to espouse the goal of developing siRNAs for the entire human genome. To achieve this goal, Cenix developed design parameters to create more efficacious siRNAs and high-throughput screening methods to enable the rapid validation of siRNA activity. The partnership of Cenix BioScience and Ambion will result in a continuous production of new validated siRNAs for the human genome and ensure that these siRNAs are produced with the highest quality standards for the entire research community.

Creating Ru les for siRNA Design
On average, 50% of the siRNAs featuring the Tuschl design rules (3' UU overhangs, <50% G/C content; 2-4) provide at least 50% reduction in target gene expression. An early goal for Cenix was to improve the percentage of effective siRNAs by improving the design rules for siRNAs. To accomplish this, Cenix tested several hundred siRNAs targeting a number of endogenous human gene products that were expressed at detectable levels in several different cell lines. The effective and ineffective siRNAs were used to judge the impact of a number of physical characteristics on siRNA activity, including Tm, predicted RNA secondary structure, nucleotide position effects, nucleotide content of the 3' overhangs, siRNA length, and nucleotide distribution over the length of the siRNA. Those characteristics that correlated with siRNA effectiveness were incorporated into a design algorithm. The final step of designing the algorithm was a BLAST search to ensure that the siRNA was specific to the gene of interest.

The algorithm was then tested for its capacity to identify effective siRNAs. siRNAs targeting 60 human genes were designed, prepared, and tested across as many as four cell lines. Of the genes whose expression could be reduced without causing cell death, 89% of the designed siRNAs provided greater than 70% reduction in target gene expression (Figure 1).

The design algorithm is now being tested on hundreds of human genes. The first siRNA sequences validated during this collaborative study are being made available by Ambion and Cenix BioScience as part of the Silencer Validated siRNA product line. As the study progresses, many more additional sequences will be added to this line.

Figure 1. Overall Performance of siRNAs Targeting Sixty Genes. siRNAs targeting 60 human genes were designed using the algorithm developed by Cenix. The top siRNA candidate for each target was prepared and transfected. Forty eight hours post-transfection, target gene expression was quantified by real-time RT-PCR. Relative reduction in mRNA expression was measured against cells transfected with a negative control siRNA. Sample size was normalized by measuring GAPDH in the various samples using real-time PCR.

Characteristics of Silencer Validated siRNAs
The average reduction in target gene expression achieved with the inital 45 Silencer Validated siRNAs tested is greater than 90% as measured by real-time RT-PCR, indicating that the design algorithm used is not only able to identify effective siRNAs at a high rate, but is also able to identify highly effective siRNAs. To further characterize the initial set of validated siRNAs, the potencies of a handful of the siRNAs were measured after transfecting HeLa cells with siRNAs at five different concentrations, ranging from 1-100 nM. As seen in Figure 2, each of the siRNAs provided a similar level of mRNA knockdown in cells transfected with 10 nM, 30 nM, and 100 nM siRNA. This confirms the potency of the siRNAs designed using the algorithm developed by Cenix, as well as the quality of the synthesis and purification processes being employed by Ambion for the Silencer Validated siRNAs. The siRNAs targeting survivin, CDK2, and GAPDH were tested further by transfecting HepG2, SK-N-AS, and MCF7 cells with five different concentrations of the siRNAs. Even in these more difficult to transfect cells, the Silencer Vali dated siRNAs were effective when transfected at concentrations below 100 nM, though the data point to the fact that poor transfection efficiency can be overcome by using higher concentrations of siRNA (Figure 3).

Figure 2. Effectiveness of Silencer Validated siRNAs at Various Concentrations. The indicated siRNAs were mixed with siPORT Lipid (Ambion) and the resulting complexes were added to HeLa cells in 24 well plates at the final concentration of siRNA shown. Forty-eight hours after transfection, RNA from the treated cells was recovered using the RNAqueous-MAG Total RNA Isolation Kit (Ambion) and reverse transcribed using the RETROscript Kit (Ambion). Target cDNA levels were measured by real-time PCR using SYBR Green assays. The expression of the target genes in the transfected cells was compared to cells transfected with an equal concentration of the Silencer Negative Control #1. Input cDNA in the different samples was normalized using real-time data for 18S rRNA. The bar graphs represent an average of three data points.

Figure 3. siRNA Activity in Various Cell Types. HeLa, HepG2, MCF7, and SK-N-AS cells in 24 well plates were transfected with the indicated siRNAs at various concentrations. Forty-eight hours after transfection, RNA from the samples was isolated using the RNAqueous-MAG Total RNA Isolation Kit (Ambion) and target gene expression was measured by real-time RT-PCR using SYBR green assays. The expression of the target genes in the transfected cells was compar ed to cells transfected with an equal concentration of the Silencer Negative Control #1 (Ambion). Input cDNA in the different samples was normalized using real-time data for 18S rRNA. The bar graphs represent the average of two data points per siRNA concentration.

Advantages of Using Highly Effective siRNA Sequences
The use of highly effective siRNAs, such as those included in the Silencer Validated siRNA product line, has a number of advantages over using moderately active siRNAs:

* It's possible to transfect less siRNA than what is traditionally used (e.g., 100 nM) and still see the same effect. You can, therefore, perform more experiments with a given amount of siRNA.

* Because less siRNA is needed, experiments can be designed that use multiple siRNAs transfected into the same cell sample without causing cytotoxicity (e.g., toxicity is usually a problem when transfecting cells with greater than 200 nM siRNA).

* Most importantly, using sequences that have been verified to effectively induce gene silencing avoids the need to screen siRNAs, and thus saves time and money, allowing the researcher to focus on biological questions. An example of one type of experiment that is possible with the Silencer Validated siRNAs is described in "Using Validated siRNAs in Functional Genomics Assays". Of course, many other applications exist, including target validation studies and pathway analysis.

* A single, validated siRNA does not require experiments to determine which siRNA is the functional siRNA of a mixture and permits e asy scale-up for subsequent experiments in animals.

The following list includes some of the genes to which our initial offering of Silencer Validated siRNAs will be targeted. This list is subject to change. We are adding new Silencer Validated siRNAs on a continual basis. For an up-to-date list and to view additional information including full gene name, accession number, and knockdown efficiency of each validated siRNA, see

AKT1 APC1 Cyclin D1 Cyclin E1 ELK-1 ERBB-2 GSK-3b PCNA PKR uPAR

SYBR is a registered trademark of MolecularProbes.

Ambion is a licensed supplier of siRNA.

Designs for the Silencer Validated siRNAs are owned by Cenix and licensed to Ambion.

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