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DNA Methylation Analysis from Body Fluids

  • Dimo DietrichEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1655)

Abstract

Circulating cell-free DNA (ccfDNA) can be found in various body fluids, i.e., blood (serum and plasma), urine, pleural effusions, and ascites. While ccfDNA predominantly originates from physiological processes, a fraction might be related to pathological events, e.g., cancer. Aberrant DNA methylation, which is considered a hallmark of cancer, can be assessed accurately in ccfDNA. Consequently, DNA methylation testing in body fluids represents a powerful diagnostic tool in the clinical management of malignant diseases. Frequently, however, the total amount of disease-related ccfDNA in a sample is low and masked by an excess of physiological ccfDNA. Thus, DNA methylation analysis of tumor-derived DNA is challenging, and high volumes of body fluids need to be analyzed in order to ensure a sufficient abundance of the analyte in the test sample. DNA methylation assays are usually based on prior conversion of cytosines to uracils by means of bisulfite. This reaction takes place under harsh chemical conditions leading to DNA degradation and therefore necessitates a proper DNA purification before downstream analyses. This article describes a protocol which allows for the preparation of ultra-pure bisulfite-converted DNA from up to 3 ml blood plasma and serum, which is well suited for subsequent molecular biological techniques, e.g., methylation-specific real-time PCR.

Key words

DNA methylation Circulating cell-free DNA ccfDNA Biomarker Plasma Serum Blood Body fluid Bodily fluid Bisulfite Magnetic beads Ammonium bisulfite 

Notes

Acknowledgment

Conflict of Interest: Dimo Dietrich is a consultant for AJ Innuscreen GmbH (Berlin, Germany) and receives royalties from product sales (innuCONVERT kits).

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Institute of PathologyUniversity Hospital of BonnBonnGermany
  2. 2.Department of Otolaryngology, Head and Neck SurgeryUniversity Hospital BonnBonnGermany

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