Comparison of Nucleosomes and Quantitative PCR Using Diverse DNA Isolation Methods

Abstract

Reliable standardization for the isolation and detection methods prior to quantification of cell-free DNA are still lacking. QIAamp DNA Blood Midi Kit (Q), the NucleoSpin-Kit (MN), and the MagNA-Pure isolation system (RD) were compared on the amount of DNA isolated versus the immunological quantification of circulating nucleosomes using the Cell Death Detection ELISA plus (RD). Forty-four plasma samples were used with each system in parallel at laboratories in Berlin and Munich. Subsequently, DNA was quantified by real-time PCR on a LightCycler 480 (RD) in Berlin using targets for ß-globin. Correlations of plasma nucleosomes and cell-free DNA measured by real-time PCR ranged between R = 0.32 and R = 0.64 and were comparable between the two laboratories. Large differences in the amounts of cell-free DNA were observed between the diverse isolation methods: the RD isolation system gave the highest DNA yield followed by MN and Q systems. Most comparable results with nucleosome ELISA were achieved using the Roche system.