Global Characterization of Circulating Nucleic Acids

Circulating nucleic acids (CNAs) include genomic and mitochondrial DNA fragments, small RNAs, and bacterial and viral DNA/RNA. Different mechanisms such as cell apoptosis, necrosis, and active CNA release from cells have been proposed to result in nucleic acids in the circulation. Application of next generation sequencing technology demonstrated that CNAs contain specific mutations, indels, microsatellite alterations, and epigenetic changes (DNA methylation) associated with various diseases. Their clinical implications have been demonstrated for diseases such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. Thus, CNAs in blood represent an attractive family of molecules that can serve as biomarkers and the analysis of CNAs can be alternative for immunohistochemical analyses of conventional biopsies. The methods described in this chapter provides details for circulating DNA and small RNA isolation, CNA(-derived cDNA) library preparation, and sequencing data analysis.