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Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success

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
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