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

  • Luca Sigalotti
  • Alessia Covre
  • Francesca Colizzi
  • Elisabetta FrattaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1909)


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.

Key words

Circulating cell-free DNA DNA methylation Bisulfite conversion Quantitative methylation-specific PCR Plasma 


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Luca Sigalotti
    • 1
  • Alessia Covre
    • 2
  • Francesca Colizzi
    • 3
  • Elisabetta Fratta
    • 3
    Email author
  1. 1.Istituto di Patologia Clinica, Azienda Sanitaria Universitaria IntegrataUdineItaly
  2. 2.Center for Immuno-Oncology, Division of Medical Oncology and Immunotherapy, Department of OncologyUniversity Hospital of SienaSienaItaly
  3. 3.Immunopathology and Cancer Biomarkers, Department of Translational ResearchCentro di Riferimento Oncologico di Aviano (CRO) IRCCSAvianoItaly

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