( Although dissociated single strand...)Visualizing siRNA in Mammalian Cells: Fluorescence Analysis of the RNAi Effect

Although dissociated single stranded siRNA was detected within the cell, the majority of the transfected siRNA appears to remain in a double stranded state. Perhaps the extent of strand separation is proportional to the molar amount of siRNA required to direct cleavage of the target gene.

3. Duration
Data presented here suggests siRNA is maintained in cells up to 10 days and that siRNA is transferred to daughter cells. If this is true and siRNA amplification does not occur in mammalian cells, then the duration of the RNAi effect should be directly proportional to the cellular concentration of the siRNA and the number of cellular divisions that occur. This suggests that a major experimental variable in siRNA experiments is the effectiveness of siRNA delivery and the initial cellular siRNA concentration.

In a situation in which an effective siRNA is transfected with low efficiency, labeled siRNA can be used to identify individual cells that have been successfully transfected with siRNA. Cells lacking labeled siRNA can thus serve as internal negative controls. Labeled siRNAs can also be used for metabolic studies, especially if performing experiments geared towards identifying clinically important molecules.

Materials and Methods
Labeling siRNA
Chemically synthesized siRNA (Ambion) was labeled using the Silencer Cy3 and FAM siRNA Labeling Kits (Ambion). Briefly, 5 g of a
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