Quantitative methylation-specific PCR

A simple method for studying epigenetic modifications of cell-free DNA

Luca Sigalotti, Alessia Covre, Francesca Colizzi, Elisabetta Fratta

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages137-162
Number of pages26
DOIs
Publication statusPublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1909
ISSN (Print)1064-3745

Fingerprint

Epigenomics
Methylation
DNA Methylation
Polymerase Chain Reaction
DNA
DNA Contamination
Tumor Biomarkers
Costs and Cost Analysis
Sensitivity and Specificity

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 Methods in Molecular Biology (pp. 137-162). (Methods in Molecular Biology; Vol. 1909). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8973-7_11

Quantitative methylation-specific PCR : A simple method for studying epigenetic modifications of cell-free DNA. / Sigalotti, Luca; Covre, Alessia; Colizzi, Francesca; Fratta, Elisabetta.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 137-162 (Methods in Molecular Biology; Vol. 1909).

Research output: Chapter in Book/Report/Conference proceedingChapter

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 Methods in Molecular Biology. Methods in Molecular Biology, vol. 1909, Humana Press Inc., pp. 137-162. https://doi.org/10.1007/978-1-4939-8973-7_11
Sigalotti L, Covre A, Colizzi F, Fratta E. Quantitative methylation-specific PCR: A simple method for studying epigenetic modifications of cell-free DNA. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 137-162. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8973-7_11
Sigalotti, Luca ; Covre, Alessia ; Colizzi, Francesca ; Fratta, Elisabetta. / Quantitative methylation-specific PCR : A simple method for studying epigenetic modifications of cell-free DNA. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 137-162 (Methods in Molecular Biology).
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