Isolation and Analysis of Circulating Nucleic Acids: Technical Advances

Martin Horlitz QIAGEN GmbH, Germany

Abstract
Circulating DNA fragments and specific messenger RNAs originating from malignant tumors, a developing fetus, but also viral or bacterial infections are present in the cell-free nucleic acids in plasma, serum and other body fluids such as urine. Access to these nucleic acids for analysis could allow for specific detection of certain disease states based on a simple blood sample. Circulating cell-free nucleic acids show distinctive properties: They are present in plasma and serum mostly as shorter fragments of less than 500 bp or nt in size. The concentration of such circulating, cell-free DNA and RNA in a plasma or serum sample is low (≈1–100 ng/ml) compared to cellular materials and varies considerably between different individuals. Because of their fragmented nature and low concentration, circulating nucleic acids present a particular challenge for efficient extraction/purification and quantification by qPCR. We present data on solutions for the following critical problems concerning the analysis of circulating nucleic acids for research and molecular diagnostic applications:
– Pre-analytical workflow (blood processing) for analyzing circulating nucleic acids
– Optimization of ccfDNA extraction from plasma samples: low target concentrations require the efficient ccfDNA enrichment from larger sample volumes (1-5 ml)
– Use of an internal control to monitor the ccfNA extraction performance
– Avoiding qPCR Quantification bias when working with fragmented target nucleic acids
– Assessment of ccfDNA fragment size distribution in plasma samples by comparing qPCR results based on target amplicons of different sizes
Furthermore, we will present data on the extraction and qPCR-based quantification of circulating fetal DNA and mRNA from maternal plasma samples obtained at the end of the first trimester of pregnancy.