Tracing of siRNAs Inside Cells by FRET Imaging
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The structural integrity of siRNA carrying fluorescent dyes attached to its different strands can be monitored through the efficiency of fluorescence resonance energy transfer between the dyes. In this chapter, the experimental details of the construction of dye-labeled siRNA and of transfection with various formulations of siRNAs with cationic lipids, nanoparticles, and liposomes, respectively, are given. Also described are the conditions for confocal microscopy and an algorithm allowing visualization of intact siRNA after image data treatment. The method allows to compare the uptake efficiency mediated by various transfection agents, and to map the location of intact siRNA inside the cell during a timeframe corresponding to a typical knockdown experiment.
Key wordssiRNA FRET Fluorescein Tetramethylrhodamine siRNA integrity R/G ratio Confocal imaging Microinjection Transfection Nanoparticles Cationic lipids Liposomes Sterical stabilized liposomes Conventional liposomes Liposomal formulation Dual asymmetric centrifugation
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