A single molecule method to quantify miRNA gene expression

Sonal Patel: Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Joanne Garver: Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Michael Gallo: Affiliation:
Epic Therapeutics, Inc. 220 Norwood Park Norwood, MA 02062 USA
Maria Hackett: Affiliation:
Novartis Institutes for Biomedical Research 250 Massachusetts Avenue Cambridge, MA 02139 USA
Stephen McLaughlin: Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Steven R. Gullans: Affiliation:
RxGen, Inc. New Haven, CT USA
Mark Nadel: Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
John Harris: Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Duncan Whitney: Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Lori A. Neely: Affiliation:
Technology & Pre-Development Millipore Corp. 80 Ashby Rd. Bedford, MA 01730 USA

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Summary

Introduction

Deemed the “breakthrough of the year” by Science magazine in 2002, research into the biology of small RNA regulation has grown exponentially in recent years; however, the field is relatively nascent in terms of identifying and characterizing the universe of miRNAs and their expression in various biological states. According to the miRNA registry (release 6.0, www.sanger.ac.uk/Software/Rfam/mirna/index.shtml); of the 319 predicted human miRNAs the expressions of 234 have been experimentally verified by Northern blot, cloning, or microarray. Further, the total number of miRNAs within a genome is unknown. Thus, sensitive, specific, quantitative, and rapid methods for measuring the expression levels of miRNAs would significantly advance the field.

The short 21 nucleotide nature of these molecules makes them difficult to study via conventional techniques. They are not easily amplified which makes miRNA microarrays and quantitative PCR technically challenging. Despite these challenges, several groups have undertaken miRNA microarray studies to quantify miRNA gene expression. Their approaches are similar in requiring up-front enrichment for small RNAs, reverse transcription, PCR amplification, labeling, and clean-up steps. While the arrays are superior at large scale screening they lack the ability to finely discriminate expression levels and are at best semi-quantitative. Theoretically the most sensitive technique to quantify miRNAs is reverse transcription RT-PCR (real time RT-PCR). However, this method is difficult in both assay (probe) design and execution. Tissue samples must be devoid of enzyme inhibitors to enable efficient reverse transcription and amplification steps (Tichopad et al., 2004).

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Type: Chapter
Information: MicroRNAs
From Basic Science to Disease Biology
, pp. 255 - 268

DOI: https://doi.org/10.1017/CBO9780511541766.022 [Opens in a new window]

Publisher: Cambridge University Press

Print publication year: 2007

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References

Ambrose, W. P., Semin, D. J., Robbins, D. L.et al. (1998). Detection system for reaction-rate analysis in a low-volume proteinase-inhibition assay. Anal. Biochem., 263, 150–157.CrossRef Google Scholar

Anazawa, T., Matsunaga, H. and Young, E. S. (2002). Electrophoretic quantitation of nucleic acids without amplification by single-molecule imaging. Anal. Chem., 74, 5033–5038.CrossRef Google Scholar

Bennink, M. L., Schaerer, O. D., Kanaar, R. and Sakata-Sogawa, K. (1999). Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1. Cytometry, 36, 200–208.Google Scholar

Brinkmeier, M., Dorre, K., Stephan, J. and Eigen, M. (1999). Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures. Analytical Chemistry, 71, 609–616.CrossRef Google Scholar

Castro, A., and Williamson, J. G. K. (1997). Single molecule detection of specific nucleic acid sequences in unamplified genomic DNA. Anal. Chem., 69, 3915–3920.CrossRef Google Scholar

Chan, E. Y., Goncalves, N., Haeusler, R. A.et al. (2004). DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags. Genome Research, 6, 1137–1146.Google Scholar

Chirico, G., Cannone, F., Beretta, S., Baldini, G. and Diaspro, A. (2001). Single molecule studies by means of the two-photon fluorescence distribution. Microsc. Res. Tech., 55, 359–364.Google Scholar

Christensen, U., Jacobsen, N., Rajwanshi, V. K., Wengel, J. and Koch, T. (2001). Stopped-flow kinetics of locked nucleic acid (LNA)-oligonucleotide duplex formation: studies of LNA-DNA and DNA-DNA interactions. Biochem. J., 354, 481–484.Google Scholar

D'Antoni, C. M., Fuchs, M., Harris, J. L.et al. (2006). Rapid quantitative analysis using a single molecule counting method. Anal. Biochem. (In press.)Google Scholar

Dundr, M., McNally, J. G., Cohen, J. and Misteli, T. (2002). Quantitation of GFP-fusion proteins in single living cells. J. Struct. Biol., 140, 92–99.Google Scholar

Eigen, M. and Rigler, R. (1994). Sorting single molecules: applications to diagnostics and evolutionary biotechnology. Proc. Natl. Acad. Sci. USA, 91, 5740–5747.CrossRef Google Scholar

Goodwin, P. M., Ambrose, W. P. and Keller, R. A. (1996). Single molecule detection in liquids by laser induced fluorescence. Acc. Chem. Res., 29, 603–613.CrossRef Google Scholar

Haab, B. B. and Mathies, R. A. (1995). Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis. Anal. Chem., 67, 3253–3260.Google Scholar

Hesse, J., Wechselberger, C., Sonnleitner, M., Schindler, H., and Schutz, G. J. (2002). Single-molecule reader for proteomics and genomics. J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci., 782, 127–135.Google Scholar

Iorio, M. V., Ferracin, M., Liu, C. G.et al. (2005). MicroRNA gene expression deregulation in human breast cancer. Cancer Research, 65(16), 7065–7070.Google Scholar

Jett, J. H., Keller, R. A., Martin, J. C.et al. (1989). High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules. J. Biomol. Struct. Dyn., 7, 301–309.Google Scholar

Korn, K., Gardellin, P., Liao, B.et al. (2003). Gene expression analysis using single molecule detection. Nucleic Acids Res., 31(16), 1–8.CrossRef Google Scholar

Li, H., Ying, L., Green, J. J., Balasubramanian, S. and Klenerman, D. (2003). Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal. Chem., 75, 1664–1670.Google Scholar

Michael, M. Z., O'Connor, S. M., Holst Pellekaan, N. G., Young, G. P., and James, R. J. (2003). Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol. Cancer. Res., 12, 882–891.Google Scholar

Neely, L. A., Patel, S., Garver, J.et al. (2006). A single-molecule method for the quantitation of microRNA gene expression. Nature Methods, 3, 41–46.Google Scholar

Nie, S., Chiu, D. T. and Zare, R. N. (1995). Real time detection of single molecules in solution by confocal fluorescence microscpy. Anal. Chem., 67, 2849–2857.Google Scholar

Novikov, E., Hofkens, J., Cotlet, M.et al. (2001). A new analysis method of single molecule fluorescence using series of photon arrival times: theory and experiment. Spectrochim. Acta A. Mol. Biomol. Spectrosc., 57, 2109–2133.CrossRef Google Scholar

R Development Core Team (2005). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Computing.

Sambrook, J., Fritsch, E. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Schwille, P. and Kettling, U. (2001). Analyzing single protein molecules using optical methods. Curr. Opin. Biotechnol., 12, 382–386.CrossRef Google Scholar

Schwille, P., Bieschke, J. and Oehlenschlager, F. (1997). Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies. Biophys. Chem., 66, 211–228.CrossRef Google Scholar

Soini, E., Meltola, N. J., Soini, A. E.et al. (2000). Two-photon fluorescence excitation in detection of biomolecules. Biochem. Soc. Trans., 28, 70–74.Google Scholar

Soper, S. A., Davis, L. M. and Shera, E. B. (1992). Similtaneous detection of two colors by two collinear laser beams at different wavelengths. J. Opt. Soc. Am. B., 9, 1761–1769.CrossRef Google Scholar

Tichopad, A., Didier, A. and Pfaffl, M. W. (2004). Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Mol. Cell Probes, 18, 45–50.Google Scholar

Widengren, J. (1995). Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J. Phys. Chem., 99, 13 368–13 379.Google Scholar

Widengren, J., and Kask, P. (1993). Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur. Biophysics 22, 169–175.Google Scholar

Yamasaki, R., Hoshino, M., Wazawa, T.et al. (1999). Single molecular observation of the interaction of GroEL with substrate proteins. J. Mol. Biol., 292, 965–972.Google Scholar

Yanagida, T., Kitamura, K., Tanaka, H., Hikikoshi Iwane, A. and Esaki, S. (2000). Single molecule analysis of the actomyosin motor. Curr. Opin. Cell. Biol., 12, 20–25.Google Scholar

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