Superior siRNA Design Combined with High Quality Custom RNA Synthesis by Ambion
Finding a highly effective siRNA can be a time consuming and labor intensive process, particularly if you are using publicly available design tools. Ambion and Cenix BioScience have partnered to provide you with high quality, effective siRNAs so you no longer need to design your own. Cenix has developed a powerful siRNA design algorithm that yields a high percentage of potent and specific siRNA sequences, and has used this algorithm to pre-design siRNAs targeting >98% of all human, mouse, and rat genes in the RefSeq database. With a major expansion of Ambion's chemical synthesis facilities, Ambion can now synthesize these made-to-order siRNAs in a variety of scales with your choice of purification options.
Design is Key to Success
To develop a rational siRNA design algorithm, Cenix and Ambion tested multiple siRNAs targeting hundreds of different human genes. The effective and ineffective siRNA sequences were used to judge the impact of a number of siRNA characteristics on their potency, including melting temperature of the duplex, nucleotide content of the 3' overhangs 16802 Silencer Pre-designed siRNA, HPLC purified 160 nmol 16804 Silencer Pre-designed siRNA, HPLC purified, annealed 40 nmol 16806 Silencer Pre-designed siRNA, HPLC purified, annealed 160 nmol 16900 Silencer Pre-designed siRNA, PAGE purified 25 nmol 16904 Silencer Pre-designed siRNA, PAGE purified, annealed 25 nmol , and nucleotide distribution over the length of the siRNA (including 5' duplex end composition shown to be important in recent publications (1,2)) to name just a few. Those characteristics that correlated with high siRNA potency were incorporated into the algorithm. As specificity is critical, a key step in the design process is a stringent comparison of each siRNA sequence to the target organism's genome sequence to eliminate those siRNAs that have a high probability of cross reaction.
Figure 1 demonstrates the high success rate of the Cenix siRNA design algorithm. In this initial experiment, 79 siRNAs designed with the algorithm were transfected into HeLa cells and mRNA levels were analyzed by real-time RT-PCR 48 hours later to assess siRNA effectiveness. Of the siRNAs tested, 74 (94%) reduced target mRNA levels by >70%. To date, more than 900 siRNAs designed using the Cenix algorithm, targeting hundreds of individual human genes, have been functionally tested. These studies demonstrate that the algorithm is robust and that it successfully addresses gene-to-gene variability in susceptibility to RNAi.
Figure 1. The Effectiveness of Cenix Designed siRNAs. siRNAs targeting 79 human genes, designed using the Cenix algorithm, were transfected into HeLa cells at a 100 nM final concentration. Target gene expression was quantified by real-time RT-PCR 48 hours post-transfection. Relative reduction in mRNA expression was measured against cells transfected with a negative control siRNA. Sample size was normalized by measuring 18S rRNA.
Ordering Pre- designed siRNAs is Easy
To take advantage of Cenix's design algorithm for your own siRNA experiments, all you need to do is provide the RefSeq number and name of your gene of interest in an easy-to-use online order form. You can now search for your gene of interest in Ambion's extensive online database, which includes all human, mouse, and rat genes for which we have siRNAs pre-designed. If we don't have your gene of interest in our database, or if you have special siRNA design requirements, such as hairpin siRNA template designs, we will be happy to generate these siRNAs on a custom basis (please inquire for pricing). Once your gene is selected, order one, two, or three Cenix designed siRNAs -- the choice is up to you. Then choose the amount of siRNA you need (typically from 20 to 160 nmol) and how you would like it purified (standard, HPLC, or PAGE). We will then synthesize the siRNA and ship it to you, along with the sequence information.
Quality siRNA Synthesis
At Ambion, each siRNA, whether pre-designed by Cenix or designed by you, is synthesized to meet the highest quality standards. Our standard purification procedure includes column purification and yields siRNA that is typically 90% pure (guaranteed >80% pure). HPLC and PAGE purification is also available when higher purity is required. As part of our rigorous quality control procedures, MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry is used to analyze the molecular weight of each RNA oligonucleotide. In addition, all HPLC and PAGE purified oligonucleotides are analyzed for purity by capillary electrophoresis (CE) or HPLC. Finally, each annealed siRNA is analyzed by gel or capillary electrophoresis to confirm that the strands have annealed properly.
See the end of this article for pricing and synthesis options. Complete ordering information is available at www.ambion.com/siRNA.
Silencer Validated siRNAs
Functionally Proven siRNAs
Ambion and Cenix have taken the idea of pre-designed siRNAs one step further with pre-made, Silencer Validated siRNAs. These siRNAs, designed using the Cenix algorithm and targeting important human genes, have been functionally proven to reduce target gene expression by at least 70%. To validate the siRNAs, individual siRNAs were transfected into one or more cell lines at a concentration of 100 nM. mRNA levels of the target gene were assessed by real-time RT-PCR 48 hours later. To be included in this elite product line, the siRNA had to demonstrably reduce mRNA levels by >70%. Most of the Silencer Validated siRNAs reduced target gene expression by 90% or more, which is a good indicator of silencing potency.
Effectiveness of Silencer Validated siRNAs
As more is learned about siRNAs, it is becoming increasingly clear that using highly potent siRNAs that efficiently silence their target gene at low siRNA concentrations is advantageous. Transfection of low siRNA concentrations reduces the potential for off target effects or induction of the antiviral response. Figure 2 shows data from six Silencer Validated siRNAs tested at five different concentrations. In all cases, silencing efficiency at 10 nM was comparable to that achieved at 100 nM siRNA, indicating that these siRNAs are effective at low concentrations. Three of these siRNAs were tested further by transfecting HepG2, SK-N-AS, and MCF7 cells at five different concentrations (Figure 3). Even in these more difficult-to-transfect cells, the Silencer Validated siRNAs were effective at low concentrations.
Figure 2. Silencer Validated siRNAs Are Effective at Low Concentrations. The indicated siRNAs were transfected into HeLa cells using siPORT Lipid at the final siRNA concentrations shown. Forty-eight hours after transfection, RNA was recovered using RNAqueous MAG-96 RNA Isolation Kit and reverse transcribed using the RETROscript Kit. Target cDNA levels were measured by real-time PCR and compared to cells transfected with an equal concentration of Silencer Negative Control #1. cDNA was normalized using real-time data for 18S rRNA. The bar graphs represent an average of three data points.
Figure 3. Silencer Validated 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 and target gene expression was measured by real-time RT-PCR. 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 the average of two data points per siRNA concentration.
Superior Quality, Pre-made siRNAs
Each Silencer Validated siRNA strand is purified by HPLC and subjected to rigorous quality control measures. After annealing, the siRNAs are analyzed by non-denaturing gel electrophoresis to ensure proper annealing, and subjected to rigorous nuclease testing. The result is the highest quality siRNA with a sequence verified to reduce gene expression. Each Silencer Validated siRNA is supplied dried at a unit size of 5 nmol along with nuclease-free water. At press time, over 200 Silencer Validated siRNAs, targeting highly studied human genes, were available.
Silencer siRNA Libraries
Accelerate Pathway Analysis and Target Validation with siRNA Libraries
Although many researchers study genes one at a time, others analyze multiple genes and sometimes entire classes of genes simultaneously. For these researchers, we have combined pre-made Cenix designed and validated siRNAs into libraries. Ambion's first siRNA libraries include the Silencer Kinase siRNA Library and the Silencer G-protein coupled receptors (GPCR) siRNA Library. Other libraries are under development. If there is a particular library that you are interested in, send us an email at firstname.lastname@example.org.
siRNAs Targeting 597 Human Kinases
The Silencer Kinase siRNA Library includes nearly 1800 siRNAs -- three individual siRNAs targeting each of 597 human kinases. All of the siRNAs have been designed using the Cenix algorithm. In addition, 528 of the siRNAs targeting 231 of the kinases are validated and have been functionally verified to reduce target mRNA levels by >70%. Subsets of the library, divided by kinase functional class, are also available.
siRNAs Targeting 441 G-protein Coupled Receptors (GPCRs)
The Silencer GPCR siRNA Library includes three siRNAs targeting each of 441 human non-olfactory GPCRs for a total of >1400 siRNAs. Like the siRNAs in the Silencer Kinase siRNA Library, each of the siRNAs has been designed using the Cenix algorithm.
Silencer Kinase siRNA Library in Action
With libraries of functional siRNAs, you can readily screen for phenotypes resulting from gene-specific inhibition, thus accelerating pathway analysis or target identification and validation studies. Cenix recently u sed 178 siRNAs from the Silencer Kinase siRNA Library to assess the impact of reducing the expression of individual kinases on cell proliferation and mitotic index (Figure 4). As seen in this example, interfering with the expression of certain kinases has dramatic effects on cell proliferation and the mitotic index, indicating the importance of these kinases in the mammalian cell cycle. This is just one example of the types of experiments possible with libraries of highly effective siRNAs.
Figure 4. Phenotypic Screen Using Silencer Kinase siRNA Library siRNAs. HeLa cells were transfected with individual siRNAs targeting 178 different human kinases. Cell number and mitotic index were measured 48 hours post transfection. The figure depicts results from 92 of the kinase siRNAs and three control siRNAs, shown in black (1=cyclin B1 siRNA, 2=RP42 siRNA, and 3=scrambled control siRNA). The gray shading indicates the normal expected ranges of cell number (top) and mitotic index (bottom).
The Silencer Kinase siRNA Library and Silencer GPCR siRNA Library include one nanomole of each siRNA in solution at a concentration of 20 M, providing sufficient material for approximately 400 transfections (100 l transfections at 25 nM siRNA) per siRNA. The siRNAs are arrayed into 96 well plates. For the kinase siRNA library, siRNAs are grouped by kinase functional class, and gene knockdown data is provided for each validated siRNA.
The pre-designed, validated and library siRNA sequences mentioned in this article are owned by Cenix and licensed exclusively to Ambion. Ambion and Cenix do not retain rights to any discoveries made using these reagents when the reagents are used as a research tool.
Ambion has been granted rights by the Massachusetts Institute of Technology to US Patent Applications 60/265232, 09/821832 and PCT/US01/10188, RNA Sequence-Specific Mediators of RNA Interference.
Cat# Product Name Size 16700 Silencer Pre-designed siRNA, standard purity 20 nmol 16702 Silencer Pre-designed siRNA, standard purity 40 nmol 16704 Silencer Pre-designed siRNA, standard purity, annealed 20 nmol 16706 Silencer Pre-designed siRNA, standard purity, annealed 40 nmol 16800 Silencer Pre-designed siRNA, HPLC purified 40 nmol