Matched siRNAs and Assays Ambion + Applied Biosystems = RNAi Success
|Ambion and Applied Biosystems have joined forces to provide a complete convenient, solution for performing gene silencing experiments and validating the results by real-time RT-PCR. Ambion's Silencer™ Validated and Silencer Pre-designed siRNAs eliminate the guesswork--and the tedious labwork--associated with siRNA design and testing. Applied Biosystems' gene-specific, ready-to-run TaqMan® Gene Expression Assays provide you with a fast, simple way to measure the efficacy of mRNA knockdown.|
Ambion's Silencer™ Pre-designed siRNAs for >34,000 human, mouse, and rat genes in the NCBI RefSeq database eliminate costly and time-consuming siRNA design, synthesis, and testing steps. Similarly, Applied Biosystems offers TaqMan® Gene Expression Assays for quantitative gene expression analysis of >41,000 human, mouse, and rat genes by real-time PCR. These assays can be used to measure mRNA knockdown for siRNA validation, to optimize transfection, and to correlate phenotype with the extent of knockdown induced by a particular siRNA.
Assays to Monitor Gene-specific siRNA Effects
Advantages of TaqMan Gene Expression Assays
Ambion, and partner Cenix BioScience, chose qRT-PCR to test Ambion's Silencer Validated siRNAs and to systematically validate more than 1100 siRNAs to verify the effectiveness of Cenix's siRNA design algorithm (this algorithm was used to design all of Ambion's Silencer Validated and Pre-designed siRNAs; see siRNA Design: It's All in the Algorithm). Figure 1 illustrates the workflow used to validate siRNAs. Using this method, siRNAs that are determined to reduce their target mRNA level by 70% or more are considered "validated" and are subsequently made available as Silencer Validated siRNAs. Figure 2 shows a small subset of validation data generated at Ambion using TaqMan Gene Expression Assays to quantitate target mRNA levels.
Figure 1. Validation of Ambion's Silencer™ Validated siRNAs. The following procedure is used by both Cenix and Ambion to validate siRNAs:
1) Cells are plated in 96 well plates and grown for 24 hours.
2) Gene specific and negative control siRNAs are independently transfected in triplicate.
3) 48 hours later, RNA is extracted.
4) Target mRNA levels are quantitated by real-time PCR.
5) Data are normalized using 18S rRNA levels.
6) The extent of target gene knockdown is expressed as a percent of mRNA remaining in cells treated with the gene-specific siRNA compared to cells treated with a negative control siRNA (Silencer Negative Control siRNA #1).
Figure 2. Silencer ™ siRNA Validation Data Generated Using Applied Biosystems TaqMan® Gene Expression Assays. The indicated Silencer Validated siRNAs were transfected into HeLa Cells at 30 nM. RNA was isolated 48 hours later and analyzed by one-step qRT-PCR using the appropriate TaqMan Gene Expression Assay (results were normalized for input RNA amount using real-time data for 18S rRNA). The inset graphs show the reduction in target gene expression compared to cells transfected with an equal concentration of Silencer Negative Control #1.
Optimizing siRNA Transfection
The Silencer GAPDH siRNA Control--and the TaqMan Gene Expression Assay for GAPDH--provide the ideal control siRNA and assay for optimization of transfection, respectively, (Figure 4).
Correlating Phenotype with the Extent of Knockdown
An example in which the phenotypic effects of an siRNA were correlated with the extent of mRNA knockdown induced is shown in Figure 5. A Silencer Validated siRNA targeting survivin mRNA was transfected into cells. Silencer Negative Control #1 siRNA was transfected into another set of cells. qRT-PCR using a TaqMan Gene Expression Assay showed that the survivin siRNA reduced survivin mRNA levels in these cells by 80% compared to cells treated with the negative control siRNA (Figure 5). In addition, immunofluorescence analysis of survivin protein levels indicated that protein levels were reduced 76% compared to cells transfected with the negative control siRNA (data not shown). In the survivin siRNA treated samples, distinct changes in nuclear morphology, consistent with changes that would be expected for cells undergoing apoptosis, were noted. No distinguishable change in nuclear morphology was noted in cells treated with the negative control siRNA as compared to nontransfected cells. From these data, it can be inferred that knockdown of survivin induces apoptosis in these samples. The next experimental step would be to confirm the results with a second siRNA targeting survivin, and to monitor apoptosis by additional assays (e.g., caspase activity assay, annexin V assay, etc.), while continuing to monitor the extent of survivin mRNA knockdown by qRT-PCR.