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

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 degradat...

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Main Authors:	Chen, Antony K., Behlke, Mark A., Tsourkas, Andrew
Format:	Online
Language:	English
Published:	Oxford University Press 2007
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018645/

id	pubmed-2018645
recordtype	oai_dc
spelling	pubmed-20186452007-10-23 Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells Chen, Antony K. Behlke, Mark A. Tsourkas, Andrew Methods Online 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. Oxford University Press 2007-08 2007-08-15 /pmc/articles/PMC2018645/ /pubmed/17702767 http://dx.doi.org/10.1093/nar/gkm593 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ 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.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Chen, Antony K. Behlke, Mark A. Tsourkas, Andrew
spellingShingle	Chen, Antony K. Behlke, Mark A. Tsourkas, Andrew Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
author_facet	Chen, Antony K. Behlke, Mark A. Tsourkas, Andrew
author_sort	Chen, Antony K.
title	Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
title_short	Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
title_full	Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
title_fullStr	Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
title_full_unstemmed	Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
title_sort	avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
description	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.
publisher	Oxford University Press
publishDate	2007
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018645/
_version_	1611404239649636352

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

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