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Confirming RNAi Effects Is Now Easier Than Ever


To validate any RNAi experiment, target mRNA reduction should be confirmed and quantitated. Validation, using a positive control siRNA that is known to be effective, is required for optimization of transfection parameters. Validation is also necessary for assessment of the effectiveness and potency of any siRNA sequence. Finally, target mRNA levels after siRNA delivery should be quantitated so that phenotypes can be correlated with the extent of mRNA knockdown.

Several different methods can be used to monitor target mRNA levels following siRNA transfection, including Northern analysis, nuclease protection assay, gel-based RT-PCR, and real-time PCR. Of these methods, real-time PCR has distinct advantages: it is the most sensitive of all of the RNA detection methods, and it can be completed in the least amount of time if optimized primer and probe sets are used.

For real-time PCR analysis of target mRNA levels for RNAi experiments, Ambion scientists recommend TaqMan Gene Expression Assays (formerly called Assays-On-Demand) from Applied Biosystems. The TaqMan Gene Expression Assays include an extensive collection of premade TaqMan primer and probe sets for quantitative, real-time PCR gene expression studies. They are considered the gold standard for gene expression analysis. Pre-designed TaqMan Gene Expression Assays are available for the human, mouse, and rat genomes, complementing Ambion's Silencer Pre-designed, Validated an
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Related biology technology :

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