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Urine of prostate cancer (PCa) carries miRNAs originated from prostate cancer cells as a part of both nucleoprotein complexes and cell-secreted extracellular vesicles. The analysis of such miRNA-markers in urine can be a convenient option for PCa screening. The aims of this study were to reveal miRNA–markers of PCa in urine and design a robust and precise diagnostic test, based on miRNA expression analysis. The expression analysis of the 84 miRNAs in paired urine extracellular vesicles (EVs) and cell free urine supernatant samples from healthy donors, patients with benign and malignant prostate tumours was done using miRCURY LNA miRNA qPCR Panels (Exiqon, Denmark). Sets of miRNAs differentially expressed between the donor groups were found in urine EVs and urine supernatant. Diagnostically significant miRNAs were selected and algorithm of data analysis, based on expression data on 24-miRNA in urine and obtained using 17 analytical systems, was designed. The developed algorithm of data analysis describes a series of steps necessary to define cut-off values and sequentially analyze miRNA expression data according to the cut-offs to facilitate classification of subjects in case/control groups and allows to detect PCa patients with 97.5% accuracy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]