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

relative GAPDH mRNA levels. C. HeLa S3 cells grown on cover slips in a 24-well dish were transfected with siRNA to the 3' UTR of GAPDH. After 4 hours, 24 hours, 3, 6, 10 and 12 days following transfection, the cells were harvested and the GAPDH protein expression was analyzed using immunofluorescence. DAPI (blue) stains cell nuclei; an antibody to GAPDH (green) binds to expressed protein.

Figure 6. Distribution of siRNA in Dividing HeLa S3 Cells. HeLa S3 cells grown on cover slips were transfected with Cy3 labeled-siRNA (red) and analyzed using immunofluorescence. Dividing cells that contained siRNA were detected in a population of cells on cover slips by observing chromosome condensation and nuclear separation/reformation. DNA was analyzed using DAPI. In all cases where the cell were dividing or appeared to have just undergone a division event the siRNA was localized to the central region of the cell.

Figure 7. siRNA Associated with Nuclei of Dividing Cells. HeLa-S3 cells grown on cover slips were transfected with FAM labeled siRNA to GAPDH. The cells were harvested and fixed using 4% Paraformaldehyde at 48 hours post transfection. The cells were mounted on microscope slides using VectaShield with DAPI and examined using the appropriate fluorescence filters. The siRNA (green) can be seen associated with each nuclei (blue) of a dividing cell.


The localization of transfected siRNA into discrete foci raised the question of whether the siRNA was being concentrated in lipid vesicles derived from the transfection
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