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Although the Silencer™ siRNA Labeling Kits were developed for labeling siRNA, they can be readily adapted for labeling long dsRNA. The following protocol can be used for this application. See "Visualizing Introduced dsRNA in Cells" for experimintal data using dsRNA labeled in this manner.
Note: Limit the exposure to light for entire procedure.
- Add the following reagents in order:
20 µl nuclease-free water
5 µl 10X Labeling Buffer
20 µl dsRNA*
5 µl Labeling Dye
50 µl Total - Incubate at 37°C for 1 hour.
- Add 5.0 µl (0.1 vol) 5 M NaCl.
- Add 125 µl (2.5 vol) cold 100% EtOH and mix thoroughly.
- Incubate at -20°C for 20-30 min.
- Centrifuge at top speed at 4°C for 20 min.
- Remove supernatant and wash pellet with 100 µl 70% EtOH.
- Centrifuge at top speed for 5 min at room temperature.
- Remove supernatant.
- Air dry pellet.
- Suspend labeled RNA in 20 µl (original volume) of nuclease-free water.
*We have successfully labeled 40 to 80 pmol of dsRNA, 400 to 1200 nt in length, with this protocol. The amount of dsRNA per reaction may need to be optimized according to the size and concentration of the dsRNA. In general, we obtain a maximum silencing effect with a final concentration of 10 nM labeled dsRNA.
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, Cy™3, to long dsRNA prepared with the MEGAscript RNAi Kit and then compared labeled dsRNA activity to that of unlabeled.
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. The specific silencing of hrp48 and U2AF50 proteins was also confirmed by immunofluorescence microscopy. 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. 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.
TOPThe 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. The specific silencing of hrp48 and U2AF50 proteins was also confirmed by immunofluorescence microscopy. 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. 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.
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