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Nucleic Acids Research Advance Access originally published online on May 17, 2008
Nucleic Acids Research 2008 36(11):e65; doi:10.1093/nar/gkn299
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Nucleic Acids Research, 2008, Vol. 36, No. 11 e65
© 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

Electrochemical detection of low-copy number salivary RNA based on specific signal amplification with a hairpin probe

Fang Wei1, Jianghua Wang2,3, Wei Liao2,3, Bernhard G. Zimmermann2,3, David T. Wong2,3 and Chih-Ming Ho1,4,*

1Department of Mechanical and Aerospace Engineering, 2UCLA School of Dentistry, 3Dental Research Institute and 4Center for Cell Control, University of California, Los Angeles, CA, USA

*To whom correspondence should be addressed. Tel: +310 825 9993; Fax: +310 206 2302; Email: chihming{at}ucla.edu

Received December 6, 2007. Revised April 25, 2008. Accepted April 29, 2008.

We developed a technique for electrochemical detection of salivary mRNA employing a hairpin probe (HP). Steric hindrance (SH) suppresses unspecific signal and generates a signal-on amplification process for target detection. The stem-loop configuration brings the reporter end of the probe into close proximity with the surface and makes it unavailable for binding with the mediator. Target binding opens the hairpin structure of the probe, and the mediator can then bind to the accessible reporter. Horseradish peroxidase is utilized to generate electrochemical signal. This signal-on process is characterized by a low basal signal, a strong positive readout and a large dynamic range. The SH is controlled via hairpin design and electrical field. By applying electric field control to HPs, the limit of detection of RNA is about 0.4 fM, which is 10 000-fold more sensitive than conventional linear probes. Endogenous Interleukin-8 mRNA is detected with the HP, and good correlation with the quantitative PCR technique is obtained. The resultant process allows a simple setup and by reducing the number of steps it is suited for the point-of-care detection of specific nucleic acid sequences from complex body fluids such as saliva.


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