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.
The ultimate goal of siRNA experiments is to assign gene function, analyze biological pathways, or validate potential drug targets. But before that goal can be realized, the siRNA has to be designed, synthesized, delivered to cells using an optimized protocol, and proven effective. Finally, it is necessary to correlate any induced phenotypic change with the extent of knockdown induced by the siRNA.

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

siRNAs exert their effects at the mRNA level. Therefore, the preferred assay for siRNA validation and transfection optimization purposes is one that quantitates target mRNA levels. Once these preliminary studies are complete, there is an advantage to measuring both target mRNA and corresponding protein levels to correlate phenotypic changes--monitored by enzymatic assay, cell based assay, gene expression profiling, or other means--with extent of knockdown induced by an siRNA.

Advantages of TaqMan Gene Expression Assays

qRT-PCR provides several advantages for monitoring target mRNA levels in RNAi experiments. It is thousands of times more sensitive than Northern analysis, results can be obtained much more quickly (assays complete in only a few hours), and the method provides quantitative results. When used with Applied Biosystems TaqMan Gene Expression Assays--off-the-shelf, pre-designed and pre-optimized primer-probe sets available for >21,000 human, >14,000 mouse, and >4,000 rat genes--the method requires no optimization. This makes real-time PCR easier to perform and the data obtained more reproducible with less signal variance than Northern analysis.

siRNA Validation

Gene silencing experiments require siRNAs that efficiently knock down expression of the target gene. To prove that a particular siRNA sequence is effective, the siRNA needs to be functionally tested in cells and be proven, or "validated," to reduce target mRNA levels by a predetermined amount. Functional testing of a large number of siRNAs designed using a particular siRNA design algorithm is also necessary to prove that the algorithm used to design the siRNA accurately predicts effective siRNAs.

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

Before siRNA experiments can be conducted to study gene function, analyze biological pathways, or validate drug targets, optimized siRNA delivery conditions must be identified and/or verified. The best way to test, optimize, and validate siRNA transfection or electroporation conditions is to use a highly effective, verified siRNA to an appropriate target and to monitor decreases in mRNA levels induced by delivery of the siRNA. Ambion uses qRT-PCR as the method of choice, and Applied Biosystems TaqMan Gene Expression Assays provide validated, easy-to-use primer-probe sets for this purpose (Figures 3 and 4).

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).

Figure 3. Overview of the Process for siRNA Transfection Optimization.


Figure 4. Using qRT-PCR to Optimize Transfection of siRNA. Cos-7 cells were transfected with CDK2 siRNA or a negative control siRNA using the indicated volumes of transfection agent per well. 48 hr after transfection, the cells were harvested and analyzed by real-time RT-PCR using TaqMan Gene Expression Assays for CDK2 and 18S rRNA. Data from the 18S rRNA reactions were used to normalize input RNA, and the percent CDK2 expression was calculated as the amount of gene expression compared to the negative control siRNA.

Correlating Phenotype with the Extent of Knockdown

To better evaluate functional siRNA experimental results, the phenotype elicited should be correlated with the extent of knockdown induced by a particular siRNA. For a complete picture, both target mRNA levels and corresponding protein levels should be analyzed. Protein levels can be monitored by Western blot, immunofluorescence, ELISA or other means. (Protein can be isolated using Ambion's PARIS™ Kit.) For quantitating mRNA levels, however, qRT-PCR is again the preferred choice.

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.

Figure 5. Knockdown of Survivin with a Silencer™ Validated siRNA and Verification with TaqMan Gene Expression Assays. HeLa cells were transfected with 30 nM of a Silencer Validated siRNA targeting survivin (BIRC5) or Silencer Negative Control #1 in quadruplicate. Forty-eight hours later, two sets of samples were stained with DAPI and analyzed by fluorescence microscopy. Total RNA was isolated from the other two sample sets using RNAqueous MAG-96 and then converted to cDNA. Target cDNA levels were analyzed in duplicate using a TaqMan Gene Expression Assay for survivin (Hs00153353_m1). Data shown here represent duplicate assays from single transfections. Survivin mRNA levels were reduced 80% in the survivin siRNA treated cells relative to control siRNA treated cells. Knockdown of survivin induced morphological changes to cell nuclei. The insets show representative cells from the survivin and control siRNA treated samples.

Summary

Thanks to optimized siRNA and real-time assay design using sophisticated bioinformatics and design algorithms, performing gene silencing experiments is now easier than ever. Pre-designed siRNA and qRT-PCR assay combinations are linked through Ambion's website. The siRNA Database provides information about Ambion's Silencer Pre-designed siRNAs targeting almost all human, mouse, and rat genes in NCBI's RefSeq database. The siRNA database is also a conduit to Silencer Validated siRNAs, which have been functionally proven to reduce target mRNA levels by >70%.