Abstract
RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy1. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents2. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage5 and engineered an improved 5′-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)–based6 signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement7. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in ≥20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.
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Acknowledgements
We thank Epoch Biosciences for the gift of FRET oligonucleotides (Redmond Red and Eclipse Quencher dyes), Kafryn W. Lieder for background materials and critical revisions, and Mary Ann Brow, James Dahlberg, and Lloyd Smith for insightful scientific discussions and revisions. This work was supported by Grant DE-FG02-94ER81891 from the Department of Energy to M. Brow, Grant 2 R44 GM57711-02A1 from the National Institutes of Health to H.S.I., and Cooperative Agreements 70NANB5H1030 and 70NANB7H3015 from the National Institute of Standards and Technology to L. Fors. M. Brow is the director of intellectual property, and L. Fors is the CEO, of Third Wave Technologies, Inc.
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Eis, P., Olson, M., Takova, T. et al. An invasive cleavage assay for direct quantitation of specific RNAs. Nat Biotechnol 19, 673–676 (2001). https://doi.org/10.1038/90290
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DOI: https://doi.org/10.1038/90290
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