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 The Easiest Route to Guaranteed Silencing ). 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).
