A protocol for preparing, characterizing and using three RNA-specific, live cell imaging probes: E36, E144 and F22

Abstract

This protocol outlines a methodology for the preparation and characterization of three RNA-specific fluorescent probes (E36, E144 and F22) and their use in live cell imaging. It describes a detailed procedure for their chemical synthesis and purification; serial product characterization and quality control tests, including measurements of their fluorescence properties in solution, measurement of RNA specificity and analysis of cellular toxicity; and live cell staining and counterstaining with Hoechst or DAPI. Preparation and application of these RNA imaging probes takes 1 week.

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Figure 1
Figure 2: Using LC-MS to monitor reaction progress and product purity.
Figure 3: Dye concentration dependency titration curve.
Figure 4: RNA versus DNA specificity test.
Figure 5: Live cell RNA staining and counterstaining.

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Acknowledgements

We gratefully acknowledge the support of the National Science Foundation (CHE-0449139) and for equipment grants for the NMR (MRI-0116222) and the Capillary LC-Ion Trap Mass spectrometer (CHE-0234863). Components of this work were conducted in a Shared Instrumentation Facility constructed with support from Research Facilities Improvement Grant C06 RR-16572 from the NCRR/NIH.

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Correspondence to Young-Tae Chang.

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The authors declare no competing financial interests.

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Li, Q., Chang, Y. A protocol for preparing, characterizing and using three RNA-specific, live cell imaging probes: E36, E144 and F22. Nat Protoc 1, 2922–2932 (2006). https://doi.org/10.1038/nprot.2006.484

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