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RNAi: Get the Whole Story

Mike Byrom, Ambion, Inc.


More and more researchers are conducting siRNA experiments to elucidate cellular pathways and gene function. Often the siRNA effect is only evaluated at the level of mRNA, but this approach overlooks the true subject of most siRNA studies--the biological effect caused by a reduction in the target protein. The biological manifestations of knockdown may not correlate directly with measured mRNA levels. Many proteins remain functionally active long after the corresponding mRNA has been removed. Other proteins need only be present in very small quantities to have a profound biological effect. In these situations, even a significant reduction at the mRNA level may not be effective at producing a cellular response. To fully understand the results of siRNA experiments, analyzing the knockdown of both the target mRNA and the corresponding protein is recommended.


Isolate RNA and Protein from the Same Sample
Until recently, separate samples have typically been required for isolation of high quality RNA and protein. However, when working with rare or difficult-to-obtain samples, it is sometimes impractical to isolate RNA and protein independently. In studies involving large numbers of samples, expensive reagents, or inherent variability (e.g. cell transfection), using separate experimental samples to obtain RNA and protein is not only costly and time consuming, but may also lead to inconsistent results. Ambion's

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