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Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification

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Abstract

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.

The nucleic acid sequence based amplification (NASBA) reaction taking place on top of a microarray. a: During the NASBA process, biotinylated antisense RNA is produced. b: Hybridization of these amplicons with their immobilized capture probes occurs during amplification. Capture probes are immobilized within spots of a surface-attached hydrogel, arranged in a microarray. Each spot potentially represents a different capture probe. ds cDNA doubled-stranded complementary DNA, PMMA polymethylmethacrylate

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Acknowledgement

This work was supported by the German Research Council (DFG RU 489/15-1).

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Authors and Affiliations

  1. Institute of Pathology, Freiburg University Medical Center, Breisacher Strasse 115a, 79106, Freiburg, Germany

    Andreas Mader & Axel zur Hausen

  2. Department of Obstetrics and Gynecology, Freiburg University Medical Center, Hugstetter Strasse 55, 79106, Freiburg, Germany

    Ulrike Riehle & Elmar Stickeler

  3. Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Köhler-Allee 103, 79110, Freiburg, Germany

    Andreas Mader, Thomas Brandstetter & Jürgen Rühe

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  1. Andreas Mader
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  2. Ulrike Riehle
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  5. Axel zur Hausen
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Correspondence to Jürgen Rühe.

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Mader, A., Riehle, U., Brandstetter, T. et al. Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification. Anal Bioanal Chem 397, 3533–3541 (2010). https://doi.org/10.1007/s00216-010-3892-4

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  • DOI: https://doi.org/10.1007/s00216-010-3892-4

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