Locus-specific analyses of DNA methylation patterns usually require a bisulfite conversion of the DNA, where cytosines are deaminated to uracils, while methylated and hydroxymethylated cytosines remain unaffected. The specific discrimination of hydroxymethylation and methylation can be achieved by introducing an oxidation of 5-hydroxymethylcytosines to 5-formylcytosines and subsequent bisulfite-mediated deamination of 5-formylcytosines.DNA methylation analysis of cell-free circulating DNA in liquid biopsies, i.e., blood samples (serum and plasma), urine, aspirates, bronchial lavages, pleural effusions, and ascites, is of great interest in clinical research. However, due to the generally low concentration of circulating cell-free DNA in body fluids, high volumes need to be analyzed. A reduction of this volume, e.g., by means of a polymer-mediated enrichment, is required in order to facilitate the bisulfite conversion. Further, these sample types usually contain a cellular fraction which is of additional interest and requires specific protocols for the sample preparation.Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used source for tissue-based clinical research. Due to degradation and covalent modifications of DNA in FFPE tissue samples, optimized protocols for the DNA preparation and bisulfite conversion are required.This chapter describes methods and protocols for the sample preparation and subsequent high-speed bisulfite conversion and DNA clean-up for several types of relevant samples, i.e., serum, plasma, urine, buffy coat, aspirates, sputum, lavages, effusions, ascites, swabs, fresh tissues, cell lines, FFPE tissues, and laser microdissected cells.Additionally, two real-time PCR assays for DNA quantification and quality control are described. The cytosine-free fragment (CFF) assay allows for the simultaneous quantification of bisulfite converted and total DNA and thus the determination of bisulfite conversion efficiency. The Mer9 real-time PCR assay amplifies the bisulfite converted sequence of the repetitive element Mer9 and enables the accurate quantification of minute DNA amounts, as present in microdissected cells and body fluids.
Keywords: Bisulfite conversion; Body fluids; DNA hydroxymethylation; DNA methylation; DNA quantification; Effusions; FFPE tissue; Plasma; Polymer-mediated enrichment; Serum.