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The discovery of the enormous role methylated cytosine plays in regulating gene expression has led to the development of a variety of techniques for detecting cytosine modification. A majority of these techniques are geared towards analyzing genomic DNA, which is typically available in large quantities. The concentration of cell-free DNAs (cfDNA) extracted from biological fluids including plasma, saliva, tears, or urine is relatively low and their degree of the fragmentation is high. Moreover, for noninvasive diagnostics of cancer, methylation patterns must be studied in minor cancer-specific fractions of DNA molecules substantially diluted by excess unmethylated molecules. The above limitations complicate the application of traditional techniques for cfDNA methylation analysis. In this manuscript, we review the state-of-art analysis of cfDNA methylation, hydroxymethylation, and noncanonical methylation (outside of CpG islands). The review covers methodological approaches to studying individual CpGs and genomic loci, as well as techniques for the large-scale analysis of methylation.

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of patients with breast tumors and healthy controls. Frequency of RASSF1A, Cyclin D2 and RARbeta2 methylation was detected using methylation-specific PCR in the extracellular DNA, extracted from plasma and cell-surface bound fractions of patient blood. Methylation of at least one of these genes was found in plasma of 13% patients with benign breast fibroadenoma and in 60% of breast cancer patients. Using cell-surface bound DNA as a substrate for PCR have lead to increase of gene methylation detection frequency up to 87% in fibroadenoma and 95% in breast cancer patients without false positive controls. GAPDH, RASSF8, Ki-67 RNA and 18S RNA were quantified using RT-qPCR of the extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The main part of the extracellular RNA was shown to be cell-surface bound. Results show a higher amount of RASSF8, Ki-67 RNA and 18S RNA in plasma and cell-bound fraction of patients with breast cancer compared with patients with benign tumors and healthy controls. The data indicate that the specific RNA quantification in blood plasma is valuable for discrimination between cancer and benign tumors, which can be detected with high sensitivity using analysis of methylated RASSF1A, Cyclin D2 and RARbeta2 genes in extracellular circulating DNA.

Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA. Concentration of cirDNA and methylation of RARbeta2, RASSF1A and HIC-1 gene promoters were investigated in cell-free and cell-surface-bound fractions from healthy donors, patients with breast cancer, and patients with breast fibroadenoma. Tumour development was shown to lead to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions. Analysis of RARbeta2 and RASSF1A methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women. Results of the study indicate that methylation-specific PCR of RARbeta2 and RASSF1A genes based on the total cirDNA combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood provide the sensitive and accurate detection and discrimination of malignant and benign breast tumours.