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An invasive cleavage assay for direct quantitation of specific RNAs

An Erratum to this article was published on 01 March 2002

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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Figure 1: Schematic of a biplex-format RNA invasive cleavage assay.
Figure 2: Cytochrome P450 specificity and quantitation of mRNAs in total RNA samples.
Figure 3: Real-time quantitation of HIV viral RNA.
Figure 4: Biplex RNA invasive cleavage assay and detection of human IL-8 and ubiquitin mRNA in cell lysates.

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References

  1. Ariyoshi, K. et al. Plasma RNA viral load predicts the rate of CD4 T cell decline and death in HIV-2-infected patients in West Africa. AIDS 14, 339–344 (2000).

    Article  CAS  Google Scholar 

  2. Brown, P.O. & Botstein, D. Exploring the new world of the genome with DNA microarrays. Nat. Genet. 21, 33–37 (1999).

    Article  CAS  Google Scholar 

  3. Science 291, 1145–1434 (2001).

  4. Black, D.L. Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367–370 (2000).

    Article  CAS  Google Scholar 

  5. Lyamichev, V. et al. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nat. Biotechnol. 17, 292–296 (1999).

    Article  CAS  Google Scholar 

  6. Ghosh, S.S., Eis, P.S., Blumeyer, K., Fearon, K. & Millar, D.P. Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer. Nucleic Acids Res. 22, 3155–3159 (1994).

    Article  CAS  Google Scholar 

  7. Van Arsdell, S.W. et al. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages. BioTechniques 28, 1220–1221, 1224–1225 (2000).

    Article  CAS  Google Scholar 

  8. Lyamichev, V.I. et al. Experimental and theoretical analysis of the invasive signal amplification reaction. Biochemistry 39, 9523–9532 (2000).

    Article  CAS  Google Scholar 

  9. Reynaldo, L.P., Vologodskii, A.V., Neri, B.P. & Lyamichev, V.I. The kinetics of oligonucleotide replacements. J. Mol. Biol. 297, 511–520 (2000).

    Article  CAS  Google Scholar 

  10. Hall, J.G. et al. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc. Natl. Acad. Sci. USA 97, 8272–8277 (2000).

    Article  CAS  Google Scholar 

  11. Ma, W.P. et al. RNA template-dependent 5′ nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases. J. Biol. Chem. 275, 24693–24700 (2000).

    Article  CAS  Google Scholar 

  12. Kwiatkowski, R.W., Lyamichev, V., de Arruda, M. & Neri, B. Clinical, genetic, and pharmacogenetic applications of the Invader assay. Mol. Diagn. 4, 353–364 (1999).

    Article  CAS  Google Scholar 

  13. Nelson, D.R. et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42 (1996).

    Article  CAS  Google Scholar 

  14. Blast. National Center for Biotechnology Information, National Institutes of Health (2000).

  15. Lane, M.J. et al. The thermodynamic advantage of DNA oligonucleotide 'stacking hybridization' reactions: energetics of a DNA nick. Nucleic Acids Res. 25, 611–617 (1997).

    Article  CAS  Google Scholar 

  16. Allawi, H.T., Dong, F., Ip, H.S., Neri, B.P. & Lyamichev, V.I. Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase. RNA 7, 314–327 (2001).

    Article  CAS  Google Scholar 

  17. Van Damme, J., Proost, P., Lenaerts, J.P. & Opdenakker, G. Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J. Exp. Med. 176, 59–65 (1992).

    Article  CAS  Google Scholar 

  18. Chaudhary, L.R. & Avioli, L.V. Dexamethasone regulates IL-1 beta and TNF-alpha-induced interleukin-8 production in human bone marrow stromal and osteoblast-like cells. Calcif. Tissue Int. 55, 16–20 (1994).

    Article  CAS  Google Scholar 

  19. Herbert, A. & Rich, A. RNA processing and the evolution of eukaryotes. Nat. Genet. 21, 265–269 (1999).

    Article  CAS  Google Scholar 

  20. Kaiser, M.W. et al. A comparison of eubacterial and archaeal structure-specific 5′- exonucleases. J. Biol. Chem. 274, 21387–21394 (1999).

    Article  CAS  Google Scholar 

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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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Correspondence to Peggy S. Eis.

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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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