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Tracing of siRNAs Inside Cells by FRET Imaging

  • Markus Hirsch
  • Il-Han Kim
  • Anne Järve
  • Roger Fischer
  • Michael F. Trendelenburg
  • Ulrich Massing
  • Karl Rohr
  • Mark HelmEmail author
Protocol
Part of the Neuromethods book series (NM, volume 58)

Abstract

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 words

siRNA 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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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Markus Hirsch
  • Il-Han Kim
  • Anne Järve
  • Roger Fischer
  • Michael F. Trendelenburg
  • Ulrich Massing
  • Karl Rohr
  • Mark Helm
    • 1
    Email author
  1. 1.Department of Chemistry, Institute of Pharmacy and Molecular BiotechnologyUniversity of HeidelbergHeidelbergGermany

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