Abstract
Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.
Highlights
We found a significant increase in mitochondrial DNA (mtDNA) recovery but no change to nuclear DNA (nDNA) recovery when SDS was added and found that the increase in mtDNA was independent of the SDS concentration (Fig. 2e)
Because we found that a reduced plasma volume (75 ml as opposed to 300 ml) achieved better mtDNA recovery, we wanted to determine whether a proportional reduction in reagent volumes used during extraction would impact DNA yields
Summary of ccf-DNA extraction scripts used by this study Each step in the process included solution volume(s), mix time, and mix speed variables
Summary
We tested these variables for both ccf-mtDNA and ccf-nDNA (total ccf-DNA) recovery by testing concentrations, volume, and time to confirm and refine the effects on ccf-DNA yields (Fig. 2). We wanted to determine the effect on ccf-DNA recovery when plasma volumes were decreased but extraction parameters were unchanged. Because we found that a reduced plasma volume (75 ml as opposed to 300 ml) achieved better mtDNA recovery, we wanted to determine whether a proportional reduction in reagent volumes used during extraction would impact DNA yields.
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