( pus fluorescence microscope. The two vi...)Visualizing siRNA in Mammalian Cells: Fluorescence Analysis of the RNAi Effect

pus fluorescence microscope. The two views represent different magnifications. Arrows point to red and green foci (individual siRNA sense and antisense strands). B. siRNA against the 3' UTR of c-myc labeled on the sense strand with Blackhole quencher and on the antisense strand with Cy3 were transfected into HeLa S3 cells. After 4, 24 and 48 hr following transfection cells were fixed and then analyzed using an Olympus fluorescence microscope.

In dividing cells, fluorescence was focused at the midline of division, suggesting that siRNA might divide equally between daughter cells. Although the majority of transfected Cy3-labeled antisense strand (red) and FAM labeled sense strand (green) siRNA was found in large yellow foci on the cytoplasmic side of the nuclear membrane, a small amount of red and green signal was also observed (Figure 4). Isolated red or green signal is the result of fluorescence from individual siRNA strands. This apparent strand separation is consistent with previous in vitro studies (Nykanen et al., 2001). To confirm and extend these observations, we used fluorescence resonance energy transfer (FRET) analysis to assess siRNAs in transfected cells. In this experiment, the sense strand of c-myc was labeled with Blackhole quencher 1 (IDT) and hybridized to the complementary Cy3 labeled antisense strand. In the double strand form, the Cy3 signal is quenched by the Blackhole quencher and almost no signal is observed during fluorescence microscopy. The quenched, double stranded siRNA was transfected into cells and examined for fluorescence at 4 , 24, 48, and 72 hr post transfection. As in the previously described dual label
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