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Nucleic Acids Research Advance Access published online on August 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm593
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© 2007 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

Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells

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 May 9, 2007. Accepted July 18, 2007.

There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.


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