An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

Abstract

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of non-invasive 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, while 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 to the initial procedure. Digestion conditions, liquid handling characteristics, and magnetic particle processor programming all contributed to increased recovery and 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.

Dynamic behavior of cell-free mitochondrial DNA in human saliva

“…To ensure the absence of cell and cell debris, each saliva sample was further centrifuged at 2,000 × g for 10 minutes at 4°C prior to processing. To avoid bias in mtDNA content that may occur during DNA isolation using column-based methods (43), DNA was extracted using an isolation-free lysis method, adapted from (44). 20 ul of saliva was added to 180ul of lysis buffer (500mM Tris HCL, 1% Tween 20, dH 2 0, and 20ug/ml proteinase K), for a dilution of 1:10.…”

Stress and circulating cell-free mitochondrial DNA: A systematic review of human studies, physiological considerations, and technical recommendations

“…To avoid bias in the extraction of either mtDNA or nDNA using standard DNA isolation kits ( Guo et al, 2009 ; Fazzini et al, 2018 ), lysis based methods which can be combined with DNA retrieval methods ( Kolesar et al, 2012 ) appear to be optimal. Optimized high-throughput methods have recently been reported that increase the efficiency of mtDNA recovery by 95-fold and nDNA recovery by 20-fold ( Ware et al, 2020 ).…”

Cellular Allostatic Load is linked to Increased Energy Expenditure and Accelerated Biological Aging

“…Culture media was collected and stored at -80°C until analyzed as a single batch. Total cell-free DNA (cf-DNA) was isolated from 75 µL f cell culture media using an automated, high throughput methodology previously published 105 , using the MagMAX Cell-Free DNA Isolation Kit based on MagMAX™ magnetic-bead technology (Thermo Fisher Scientific). Duplex qPCR reactions with Taqman chemistry were used to simultaneously quantify mitochondrial (cf-mtDNA, ND1) and nuclear (cf-nDNA, B2M) amplicons, using the same primers and probes described in the mtDNA copy number section.…”