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Fluorescently Labeling Long dsRNA with the Silencer siRNA Labeling Kit

l concentration of 10 nM labeled dsRNA.



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 the 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 1A 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 1A). The specific silencing of hrp48 and U2AF50 proteins was also confirmed by immunofluorescence microscopy (data not shown). Direct observation of the Cy3 fluorescence demonstrated that both hrp48 and U2AF50 labeled dsRNA were taken up equivalently into L2 cells and localized to discrete perinuclear foci in the cytoplasm (Figure 1B). This localization is very similar to what was previously reported for siRNA in mammalian cells (Byron et al., 2002). Finally time course studies showed that the RNAi effect was maintained in cultured Drosophila cells for up to 10 days and that
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