FRET imaging of cells transfected with siRNA/liposome complexes.
By monitoring the efficiency of fluorescence resonance energy transfer of dyes attached to the different strands of siRNA, the structural integrity of the latter can be traced inside cells. Here, the experimental details of dye-labeled siRNA construction, tissue culture, and transfection with liposomally formulated siRNAs are given, as well as the conditions for confocal microscopy and an algorithm allowing the visualization of intact siRNA after image data treatment. The method allows rapid screening of different liposomal siRNA formulations, obtained by small scale dual asymmetric centrifugation with high entrapping efficiency.
Kim
Il-Han
IH
Department of Bioinformatics and Functional Genomics, German Cancer Research Center, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany.
Järve
Anne
A
Hirsch
Markus
M
Fischer
Roger
R
Trendelenburg
Michael F
MF
Massing
Ulrich
U
Rohr
Karl
K
Helm
Mark
M
D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D008845:Microinjections; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000032:analysis*; D051381:Rats; D016707:Tissue Fixation; D014162:Transfection
D005456:Fluorescent Dyes; D008081:Liposomes; D034741:RNA, Small Interfering