( Although the A ...)Fluorescently Labeling Long dsRNA with the Silencer siRNA Labeling Kit

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.

1. Add the following reagents in order:

20 l nuclease-free water
5 l 10X Labeling Buffer
20 l dsRNA*
5 l Labeling Dye
50 l Total

2. Incubate at 37C for 1 hour.
3. Add 5.0 l (0.1 vol) 5 M NaCl.
4. Add 125 l (2.5 vol) cold 100% EtOH and mix thoroughly.
5. Incubate at -20C for 20-30 min.
6. Centrifuge at top speed at 4C for 20 min.
7. Remove supernatant and wash pellet with 100 l 70% EtOH.
8. Centrifuge at top speed for 5 min at room temperature.
9. Remove supernatant.
10. Air dry pellet.
11. Suspend labeled RNA in 20 l (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 fina
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