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Cell-free DNA as Diagnostic Markers pp 137–162Cite as

Quantitative Methylation-Specific PCR: A Simple Method for Studying Epigenetic Modifications of Cell-Free DNA

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1909))

Abstract

Aberrant DNA methylation of cell-free circulating DNA (cfDNA) has recently gained attention for its use as biomarker in cancer diagnosis, prognosis, and prediction of therapeutic response. Quantification of cfDNA methylation levels requires methods with high sensitivity and specificity due to low amounts of cfDNA available in plasma, high degradation of cfDNA, and/or contamination with genomic DNA. To date, several approaches for measuring cfDNA methylation have been established, including quantitative methylation-specific PCR (qMSP), which represents a simple, fast, and cost-effective technique that can be easily implemented into clinical practice. In this chapter, we provide a detailed protocol for SYBR Green qMSP analysis which is currently used in our laboratory for cfDNA methylation detection. Useful information regarding successful qMSP primers design are also provided.

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Authors and Affiliations

  1. Istituto di Patologia Clinica, Azienda Sanitaria Universitaria Integrata, Udine, Italy

    Luca Sigalotti

  2. Center for Immuno-Oncology, Division of Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Siena, Italy

    Alessia Covre

  3. Immunopathology and Cancer Biomarkers, Department of Translational Research, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    Francesca Colizzi & Elisabetta Fratta

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  1. Luca Sigalotti
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  2. Alessia Covre
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  3. Francesca Colizzi
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  4. Elisabetta Fratta
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Correspondence to Elisabetta Fratta .

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  1. Biosciences Laboratory, IRCCS I.R.S.T, Meldola, Italy

    Valentina Casadio

  2. Biosciences Laboratory, IRCCS I.R.S.T, Meldola, Italy

    Samanta Salvi

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Sigalotti, L., Covre, A., Colizzi, F., Fratta, E. (2019). Quantitative Methylation-Specific PCR: A Simple Method for Studying Epigenetic Modifications of Cell-Free DNA. In: Casadio, V., Salvi, S. (eds) Cell-free DNA as Diagnostic Markers. Methods in Molecular Biology, vol 1909. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8973-7_11

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