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Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

Nature Nanotechnology volume 8, pages 125–129 (2013)Cite this article

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Abstract

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene1,2,3. Drugs that target tumours carrying this mutation have recently entered the clinic4,5,6,7. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods8. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl–1 of RNA material, without prior PCR amplification and use of labels.

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Figure 1: Principle of microcantilever array functionalization and measurement.
Figure 2: Compressive surface stress from hybridization experiments with PCR-amplified BRAF sequences.
Figure 3: Detection of mutated versus wild-type BRAF in total RNA samples.
Figure 4: Analysis of RNA samples from different BRAF mutated and wild-type tumour cells.

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Acknowledgements

The authors acknowledge ongoing support from M. Despont and U. Drechsler (IBM Research GmbH) in providing cantilever arrays. The authors also thank the National Center of Competence for Nanoscale Science (NCCR Nano), the Swiss Nano Institute (SNI), the NanoTera Program, the Cleven Foundation and the Swiss National Science Foundation for financial support. K. Muehlethaler is thanked for excellent technical assistance.

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Author notes

  1. D. Rimoldi and Ch. Gerber: These authors contributed equally to this work

Authors and Affiliations

  1. Swiss Nano Institute, University of Basel, Klingelbergstrasse 82, Basel, 4056, Switzerland

    F. Huber, H. P. Lang, N. Backmann & Ch. Gerber

  2. Ludwig Center for Cancer Research of the University of Lausanne, Epalinges, 1066, Switzerland

    D. Rimoldi

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  1. F. Huber
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  2. H. P. Lang
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  5. Ch. Gerber
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Contributions

F.H., D.R. and C.G. conceived the study. F.H. and D.R. designed the experiments and interpreted the data. F.H. performed and analysed the experiments. D.R. prepared DNA/RNA samples and cell lines. H.P.L. gold-coated the cantilever arrays. F.H., D.R., H.P.L., C.G. and N.B. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to F. Huber.

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Huber, F., Lang, H., Backmann, N. et al. Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays. Nature Nanotech 8, 125–129 (2013). https://doi.org/10.1038/nnano.2012.263

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