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Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

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Abstract

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L−1, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.

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Acknowledgements

This work was carried out as part of the Commission of the European communities ICT project Integrated Microsystem for the magnetic isolation and Analysis of Single circulating tumour Cells for Oncology diagnostics and Therapy follow-up, MASCOT, FP6-2004-IST-026752 and partly supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme the (contract FP7-IEF-221198).

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Authors and Affiliations

  1. Nanobiotechnology and Bioanalysis Group, Departament d’Enginyería Quimica, Universitat Rovira i Virgili, 26 Avinguda Països Catalans, 43007, Tarragona, Spain

    Josep L. Acero Sanchez, Olivier Y. F. Henry, Teresa Mairal & Ciara K. O’Sullivan

  2. MRC Holland, Willem Schoutenstraat 6, 1057 DN, Amsterdam, The Netherlands

    Nadja Laddach & Anders Nygren

  3. AdnaGen AG, Ostpassage 7, 30853, Langenhagen, Germany

    Siegfried Hauch & Jasmin Fetisch

  4. Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 08010, Barcelona, Spain

    Ciara K. O’Sullivan

Authors
  1. Josep L. Acero Sanchez
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  2. Olivier Y. F. Henry
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  3. Teresa Mairal
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  4. Nadja Laddach
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  5. Anders Nygren
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  6. Siegfried Hauch
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  7. Jasmin Fetisch
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  8. Ciara K. O’Sullivan
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Correspondence to Olivier Y. F. Henry or Ciara K. O’Sullivan.

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Acero Sanchez, J.L., Henry, O.Y.F., Mairal, T. et al. Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification. Anal Bioanal Chem 397, 2325–2334 (2010). https://doi.org/10.1007/s00216-010-3830-5

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  • DOI: https://doi.org/10.1007/s00216-010-3830-5

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