Abstract
Circulating, cell-free mitochondrial DNA (ccf-mtDNA) and nuclear DNA (ccf-nDNA) are under investigation as biomarkers for various diseases. Optimal ccf-mtDNA isolation parameters, like those outlined for ccf-nDNA, have not been established. Here, we optimized a protocol for both ccf-mtDNA and ccf-nDNA recovery using a magnetic bead-based isolation process on an automated 96-well platform. Using the optimized protocol, our data show 6-fold improved yields of ccf-mtDNA when compared to the starting protocol. Digestion conditions, liquid handling characteristics, and magnetic particle processor programming all contributed to increased recovery and improved reproducibility. To our knowledge, this is the first high-throughput approach optimized for mtDNA and nDNA recovery and serves as an important starting point for clinical studies.
Abbreviations
- ccf
- circulating, cell-free;
- DWP
- deep well plate;
- ES
- Elution Solution;
- exp
- experimental value;
- h
- hours;
- LBS
- Lysis/Binding Solution;
- LiHa
- liquid handler;
- MPP
- magnetic particle processor;
- min
- minutes;
- mtDNA
- mitochondrial DNA;
- nDNA
- nuclear DNA
- ProK
- Proteinase K;
- RoMa
- robotic arm;
- RT
- room temperature;
- SDS
- sodium dodecyl sulfate;
- sec
- seconds;
- TC
- tip comb;
- qPCR
- quantitative polymerase chain reaction.