Nucleic Acids Research Advance Access published online on September 22, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm694
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Methods Online |
Surveillance of siRNA integrity by FRET imaging
1Department of Chemistry, Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany, 2Department of Bioinformatics and Functional Genomics, IPMB, University of Heidelberg, and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany, 3Department of Pharmaceutical Technology und Pharmacology, IPMB, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany, 4Tumor Biology Center, Department of Clinical Research, Breisacher Str. 117, D-79106 Freiburg, Germany, 5Department of Hygiene and Medical Microbiology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, 6German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany and 7IPMB Master Program Molecular Biotechnology
*To whom correspondence should be addressed. Tel: +49 6221 544879; Fax: +49 6221 546430; Email: mark.helm{at}urz.uni-heidelberg.de
Received May 9, 2007. Revised August 21, 2007. Accepted August 22, 2007.
Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.