RNA expression profiling at the single molecule level

  1. Jan Hesse1,
  2. Jaroslaw Jacak1,
  3. Maria Kasper2,
  4. Gerhard Regl2,
  5. Thomas Eichberger2,
  6. Martina Winklmayr2,
  7. Fritz Aberger2,
  8. Max Sonnleitner3,
  9. Robert Schlapak3,
  10. Stefan Howorka3,
  11. Leila Muresan4,
  12. Anna-Maria Frischauf2,5, and
  13. Gerhard J. Schütz1,5
  1. 1 Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz, Austria;
  2. 2 Division of Genomics, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria;
  3. 3 Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, A-4020 Linz, Austria;
  4. 4 Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, A-4040 Linz, Austria

Abstract

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration—corresponding to only 39,000 molecules in the sample solution—and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 104 cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Footnotes

  • ↵5 Corresponding authors.

    ↵5 E-mail gerhard.schuetz{at}jku.at; fax 43-732-3468-29284.

    ↵5 E-mail annemarie.frischauf{at}sbg.ac.at; fax 43-662-8044-183.

  • [Supplemental material is available online at www.genome.org.]

  • Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.4999906

    • Received December 2, 2005.
    • Accepted May 4, 2006.

  • Freely available online through the Genome Research Open Access option.

Articles citing this article

  • Ultrasensitive RNA profiling: Counting single molecules on microarrays Genome Res October 1, 2006 16: 1195-1197
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  1. Published in Advance June 29, 2006, doi: 10.1101/gr.4999906 Genome Res. 16: 1041-1045 Copyright © 2006, Cold Spring Harbor Laboratory Press

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