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Visualizing siRNA in Mammalian Cells: Fluorescence Analysis of the RNAi Effect

Mike Byrom, Vince Pallotta, David Brown*, and Lance Ford*
Ambion, Inc.
2130 Woodward Street
Austin, TX 78744-1832
* Corresponding authors dbrown@ambion.com, lford@ambion.com


Abstract
Labeling GAPDH and c-myc siRNA with either fluorescein or Cy3 was demonstrated to have no effect on silencing efficiency, but allowed visualization of the distribution patterns of these molecules after transfection. Using fluorescence microscopy we have analyzed the dynamics of siRNA localization. Our studies revealed that the majority of both GAPDH and c-myc siRNA was localized to the nuclear periphery. In addition, in vivo siRNA strand separation was observed, supporting similar observations from earlier in vitro studies (Zamore et al., 2000).

Using fluorescently labeled siRNA, the time course of gene silencing was examined at both the mRNA and protein level. GAPDH protein and mRNA levels were moderately reduced by 4 hours post transfection, but intensified to maximum levels by 24 to 48 hours post transfection. Both mRNA and protein levels remained suppressed as long as 10 days post transfection. The initiation of the silencing effect was found to correlate well with the release of siRNA from the transfection agent in the cytoplasm of transfected cells and the occurrence of strand separation during this period of time.



Introduction
Small interfering RNAs (siRNAs) can induce specific post-transcriptional
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