Quantification of MicroRNAs, Splicing Isoforms, and Homologous mRNAs With the Invader Assay

  • Peggy S. Eis
  • Mariano A. Garcia-Blanco
Part of the Methods in Molecular Biology book series (MIMB, volume 488)


The understanding of physiology and pathology requires accurate quantification of intracellular concentrations of important molecules such as unique RNA species. Accurate quantification of highly homologous messenger RNAs (mRNAs) (1, 2, 3), alternatively spliced mRNAs (4), and the short microRNAs (miRNAs) (5,6) has been successfully achieved using the Invader assay. This method directly detects specific RNA molecules in preparations of pure total cellular RNA (1– 100 ng) or in crude cell lysate (103–104 cells) samples using an isothermal signal amplification process with a fluorescence resonance energy transfer (FRET)-based fluorescence readout. Features of the Invader assay include the ability to detect 1–10 RNA molecules per cell, to discriminate between RNAs that differ by a single base, and to precisely measure 1.2-fold changes in RNA expression. Further, an isothermal format and the ability to detect two different RNA molecules with a biplex format make the Invader assay suitable for high-throughput screening applications.

Key Words:

Alternative splicing Cleavase enzyme FRET gene expression high-throughput screening HTS Invader assay invasive cleavage microRNA miRNA mRNA RNA quantifi-cation/quantitation splice variant 



We acknowledge support from National Institutes of Health grants GM 63090 (to M.A. G.-B.) and GM 30220 (to J.E. Dahlberg). We thank James Dahlberg for permission to use the miR-155 microRNA data and for critical reading of the manuscript.


  1. 1.
    Eis, P. S., Olson, M. C., Takova, T., et al. (2001) An invasive cleavage assay for direct quantitation of specific RNAs. Nat. Biotechnol. 19, 673–676.CrossRefPubMedGoogle Scholar
  2. 2.
    Burczynski, M. E., McMillian, M., Parker, J. B., et al. (2001) Cytochrome p450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay. Drug Metab. Dispos. 29, 1243–1250.PubMedGoogle Scholar
  3. 3.
    Mills, J. B., Rose, K. A., Sadagopan, N., Sahi, J., and de Morais, S. M. (2004) Induction of drug metabolism enzymes and MDR1 using a novel human hepato-cyte cell line. J. Pharmacol. Exp. Ther. 309, 303–309.CrossRefPubMedGoogle Scholar
  4. 4.
    Wagner, E. J., Curtis, M. L., Robson, N. D., Baraniak, A. P., Eis, P. S., and Garcia-Blanco, M. A. (2003) Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay. RNA 9, 1552–1561.CrossRefPubMedGoogle Scholar
  5. 5.
    Allawi, H. T., Dahlberg, J. E., Olson, S., et al. (2004) Quantitation of microRNAs using a modified Invader assay. RNA 10, 1153–1161.CrossRefPubMedGoogle Scholar
  6. 6.
    Eis, P. S., Tam, W., Sun, L., et al. (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc. Natl. Acad. Sci. U. S. A. 102, 3627–3632.CrossRefPubMedGoogle Scholar
  7. 7.
    Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.CrossRefPubMedGoogle Scholar
  8. 8.
    Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.CrossRefPubMedGoogle Scholar
  9. 9.
    Bartel, D. P., and Chen, C. Z. (2004) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet. 5, 396–400.CrossRefPubMedGoogle Scholar
  10. 10.
    Johnson, J. M., Castle, J., Garrett-Engele, P., et al. (2003) Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302, 2141–2144.CrossRefPubMedGoogle Scholar
  11. 11.
    Neville, M., Seltzer, R., Aizenstein, B., et al. (2002) Characterization of cyto-chrome P450 2D6 alleles using the Invader system. Biotechniques 32, S34–S43.Google Scholar
  12. 12.
    Ohnishi, Y., Tanaka, T., Ozaki, K., Yamada, R., Suzuki, H., and Nakamura, Y. (2001) A high-throughput SNP typing system for genome-wide association studies J. Hum. Genet. 46, 471–477.CrossRefPubMedGoogle Scholar
  13. 13.
    Lyamichev, V., Mast, A. L., Hall, J. G., et al. (1999) Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucle-otide probes. Nat. Biotechnol. 17, 292–296.CrossRefPubMedGoogle Scholar
  14. 14.
    Hall, J. G., Eis, P. S., Law, S. M., et al. (2000) Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc. Natl. Acad. Sci. U. S. A. 97, 8272–8277.CrossRefPubMedGoogle Scholar
  15. 15.
    de Arruda, M., Lyamichev, V. I., Eis, P. S., et al. (2002) Invader technology for DNA and RNA analysis: principles and applications. Expert Rev. Mol. Diagn. 2, 487–496.CrossRefPubMedGoogle Scholar
  16. 16.
    Berggren, W. T., Takova, T., Olson, M. C., Eis, P. S., Kwiatkowski, R. W., and Smith, L. M. (2002) Multiplexed gene expression analysis using the Invader RNA assay with MALDI-TOF mass spectrometry detection. Anal. Chem. 74, 1745–1750.CrossRefPubMedGoogle Scholar
  17. 17.
    Chan-Hui, P. Y., Stephens, K., Warnock, R. A., and Singh, S. (2004) Applications of eTag assay platform to systems biology approaches in molecular oncology and toxicology studies. Clin. Immunol. 111, 162–174.CrossRefPubMedGoogle Scholar
  18. 18.
    Nagano, M., Yamashita, S., Hirano, K., et al. (2002) Two novel missense mutations in the CETP gene in Japanese hyperalphalipoproteinemic subjects: high-throughput assay by Invader assay. J. Lipid Res. 43, 1011–1018.CrossRefPubMedGoogle Scholar
  19. 19.
    Ma, W. P., Kaiser, M. W., Lyamicheva, N., et al. (2000) RNA template-dependent 5 nuclease activity of Thermus aquaticus and Thermus thermophilusDNA poly-merases. J. Biol. Chem. 275, 24693–24700.CrossRefPubMedGoogle Scholar
  20. 20.
    Kaiser, M. W., Lyamicheva, N., Ma, W., et al. (1999) A comparison of eubacte-rial and archaeal structure-specific 5 - exonucleases. J. Biol. Chem. 274, 21387–21394.CrossRefPubMedGoogle Scholar
  21. 21.
    Lyamichev, V. I., Kaiser, M. W., Lyamicheva, N. E., et al. (2000) Experimental and theoretical analysis of the invasive signal amplification reaction. Biochemistry 39, 9523–9532.CrossRefPubMedGoogle Scholar
  22. 22.
    Lane, M. J., Paner, T., Kashin, I., et al. (1997) The thermodynamic advantage of DNA oligonucleotide “stacking hybridization” reactions: energetics of a DNA nick. Nucleic Acids Res. 25, 611–617.CrossRefPubMedGoogle Scholar
  23. 23.
    Majlessi, M., Nelson, N. C., and Becker, M. M. (1998) Advantages of 2 -O-methyl oligoribonucleotide probes for detecting RNA targets. Nucleic Acids Res. 26, 2224–2229.CrossRefPubMedGoogle Scholar
  24. 24.
    Olson, M. C., Takova, T., Chehak, L., et al. (2004) Invader Assay for RNA Quantitation, Humana Press, Totowa, NJ.Google Scholar
  25. 25.
    Levanon, E. Y. , Eisenberg, E., Yelin, R., et al. (2004) Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nat. Biotechnol. 22, 1001–1005.CrossRefPubMedGoogle Scholar
  26. 26.
    Bass, B. L. (2002) RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846.CrossRefPubMedGoogle Scholar
  27. 27.
    Zuker, M. (2003) Mfold Web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.CrossRefPubMedGoogle Scholar
  28. 28.
    Griffiths-Jones, S. (2004) The microRNA Registry. Nucleic Acids Res. 32, D109– D111.CrossRefPubMedGoogle Scholar
  29. 29.
    Allawi, H. T., Dong, F., Ip, H. S., Neri, B. P., and Lyamichev, V. I. (2001) Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase. RNA 7, 314–327.CrossRefPubMedGoogle Scholar
  30. 30.
    Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. (1994) Molecular Biology of the Cell, 3rd ed., Garland, New York.Google Scholar
  31. 31.
    Mathews, D. H., Burkard, M. E., Freier, S. M., Wyatt, J. R., and Turner, D. H. (1999) Predicting oligonucleotide affinity to nucleic acid targets. RNA 5, 1458–1469.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Peggy S. Eis
    • 1
  • Mariano A. Garcia-Blanco
    • 2
  1. 1.Inc., MadisonRoche NimbleGenWisconsinUSA
  2. 2.Departments of Molecular Genetics and Microbiology and of MedicineDuke University Medical CenterDurhamUSA

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