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RNAi in Non-Mammalian Cultured Cells

="http://www.ambion.com/catalog/ProdGrp.html?fkApp=5&fkSubApp=16&fkProdGrp=54">NorthernMax-Gly Kit, Ambion). Radiolabeled RNA probes were produced (MAXIscript Kit, Ambion) from two 300 bp long PCR templates (U2AF50 and hrp48) or from Ambion pTRI RNA 28S plasmid. The latter probe was used to normalize the amount of RNA added. (C) Dose sensitive reduction of hrp48 protein expression.


Visualizing Introduced dsRNA in Cells
 One way to further understand the mechanism of RNAi is to track the introduced dsRNA in live cells. Fluorescent labeling is commonly used by scientists to localize macromolecules, but it was not known whether the incorporation of a fluorophore into dsRNA would affect its ability to induce gene silencing. To address this question, we used Ambion's Silencer siRNA Labeling Kit to couple the fluorescent label, Cy3, to long dsRNA prepared with the MEGAscript RNAi Kit and then compared labeled dsRNA activity to that of unlabeled. Figure 3A shows that the same powerful silencing effect was obtained with both labeled and unlabeled dsRNA specific for hrp48 or U2AF50 gene products. No cytotoxic effect was associated with the fluorescent moiety and the expression level of a non-related control protein, Pab 1, was not affected (Figure 3A). The specific silencing of hrp48 and U2AF50 proteins was also confirmed by immunofluorescence microscopy (data not
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