Nucleic Acids Research Advance Access published online on February 14, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn039
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Methods Online |
Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons
1Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332 and 2Division of Cardiology, Emory University, Atlanta, GA 30322, USA
*To whom correspondence should be addressed. Tel: +1 404 385 0373; Fax: +1 404 894 4243; Email: gang.bao{at}bme.gatech.edu
Received September 26, 2007. Revised January 14, 2008. Accepted January 22, 2008.
The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.