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Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips

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Abstract

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (μPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the μPCR chip down to 5 starting target molecules using TaqMan ® chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 μl assays. The high sensitivity and quantification capability of the μPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

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Acknowledgments

The authors would like to thank Stephen Gelling and Liz Reusche for the assistance with the manuscript as well as Ulrich Barenbrock for his support in scientific project management. Research was supported by the Max Planck Society, the European Moltools Project and Applied Biosystems. This work is part of Andreas Dahl's PhD project.

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Authors and Affiliations

  1. Max-Planck-Institute for Molecular Genetics, Department Vertebrate Genomics, Ihnestrasse 73, 14195, Berlin-Dahlem, Germany

    Andreas Dahl, Marc Sultan, Regine Schwartz, Matthias Lange, Hans Lehrach & Lajos Nyarsik

  2. Applied Biosystems PE Deutschland GmbH, Askaniaweg 6, D-88662, Überlingen, Germany

    Alexander Jung & Michael Steinwand

  3. Applied Biosystems 850 Lincoln Centre Drive, Foster City, CA, 94404, USA

    Kenneth J. Livak

  4. Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustrasse 3, 14195, Berlin-Dahlem, Germany

    Andreas Dahl & Marc Sultan

Authors
  1. Andreas Dahl
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  2. Marc Sultan
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  3. Alexander Jung
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  4. Regine Schwartz
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  5. Matthias Lange
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  6. Michael Steinwand
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  7. Kenneth J. Livak
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  8. Hans Lehrach
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  9. Lajos Nyarsik
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Correspondence to Andreas Dahl.

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Dahl, A., Sultan, M., Jung, A. et al. Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips. Biomed Microdevices 9, 307–314 (2007). https://doi.org/10.1007/s10544-006-9034-2

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  • DOI: https://doi.org/10.1007/s10544-006-9034-2

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