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Nucleic Acids Research Advance Access originally published online on May 24, 2008
Nucleic Acids Research 2008 36(12):e69; doi:10.1093/nar/gkn331
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Nucleic Acids Research, 2008, Vol. 36, No. 12 e69
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA

Antony K. Chen1, Mark A. Behlke2 and Andrew Tsourkas1,*

1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104 and 2Integrated DNA Technologies, Inc., Coralville, IA 52241, USA

*To whom correspondence should be addressed. Tel: +215 898 8167; Fax: +215 573 2071; Email: atsourk{at}seas.upenn.edu

Received March 29, 2008. Revised May 6, 2008. Accepted May 8, 2008.

Fluorescent microscopy experiments show that when 2'-O-methyl-modified molecular beacons (MBs) are introduced into NIH/3T3 cells, they elicit a nonspecific signal in the nucleus. This false-positive signal can be avoided by conjugating MBs to macromolecules (e.g. NeutrAvidin) that prevent nuclear sequestration, but the presence of a macromolecule makes efficient cytosolic delivery of these probes challenging. In this study, we explored various methods including TAT peptide, Streptolysin O and microporation for delivering NeutrAvidin-conjugates into the cytosol of living cells. Surprisingly, all of these strategies led to entrapment of the conjugates within lysosomes within 24 h. When the conjugates were pegylated, to help prevent intracellular recognition, only microporation led to a uniform cytosolic distribution. Microporation also yielded a transfection efficiency of 93% and an average viability of 86%. When cells microporated with MB–NeutrAvidin conjugates were examined via flow cytometry, the signal-to-background was found to be more than 3 times higher and the sensitivity nearly five times higher than unconjugated MBs. Overall, the present study introduces an improved methodology for the high-throughput detection of RNA at the single cell level.


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