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Methods

RNA expression profiling at the single molecule level

¹ Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz, Austria; ² Division of Genomics, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria; ³ Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, A-4020 Linz, Austria; ⁴ Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, A-4040 Linz, Austria

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.

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

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

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