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RNAi in Non-Mammalian Cultured Cells

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 3B). 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 labeled dsRNA was passed on to daughter cells during cell division (data not shown).

Figure 3. Fluorescently Labeled dsRNA Retains Functionality. (A) Silencing experiments and Western blot analyses were performed as described in Figure 2 three days after treatment with 10 nM of the indicated labeled or unlabeled dsRNA. (B) dsRNA specific for U2AF50 was labeled with Cy3 using the Silencer siRNA Labeling Kit, introduced into Drosophila L2 cells, and the cells were analyzed by fluorescence microscopy. BLUE: DAPI stained nuclei. RED: Cy3 labeled U2AF50 dsRNA.


The Silencer Cy3 and FAM siRNA Labeling Kits can be used to label siRNA and long dsRNA, and are the perfect complement to the MEGAscript RNAi Kit. Labeled dsRNA can be used to analyze subcellular localization, stability, and uptake. In addition, fluorescently labeled dsRNA is particularly well-suited for double label experiments (with a labeled antibody) to track cells that take up dsRNA and to correlate uptake with down-regulation of the target protein. Although the Silencer
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