Comparison of methods for isolation and quantification of circulating cell-free DNA from patients with endometriosis

Hanna Huebner, Hannah Lubrich, Lubrich H, Simon Blum, S Blum, Antoniadis S, Sophia Antoniadis, Lermann J, Johannes Lermann, Arif Ekici, Arif B. Ekici, Fasching PA, Peter A. Fasching, Matthias W. Beckmann, Beckmann MW, Ruebner M, Matthias Ruebner, Stefanie Burghaus
Reproductive BioMedicine Online · 2021 · vol. 43(5) , pp. 788–798 · doi:10.1016/j.rbmo.2021.08.004 · PMID:34493460 · W3189200787
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This study compared methods for isolating and quantifying circulating cell-free DNA in endometriosis patients to determine the most effective approach.

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Abstract

Research questionWhich is the optimal extraction method for isolating and quantifying circulating cell-free DNA (ccfDNA) from patients with endometriosis? Endometriosis is a common benign disease, associated with pain, infertility and reduced quality of life. Endometriosis is also a known risk factor for various cancers. Robust biomarkers for early detection and prediction of prognosis, however, are lacking. CcfDNA is an easy to obtain biomarker associated with prognosis of cancer patients and enables non-invasive analysis of somatic mutations. Recently, elevated levels of ccfDNA were detected in patients with endometriosis.DesignTwo different ccfDNA extraction methods were compared: Maxwell RSC ccfDNA plasma kit (Maxwell) and QiAamp minElute ccfDNA mini kit (QIAamp). The ccfDNA and circulating mitochondrial DNA (mtDNA) quantities from 34 patients diagnosed with endometriosis were analysed. Fluorometric measurement and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of short and long ALU and mtDNA fragments were used to quantiy ccfDNA.ResultsThe yield of ccfDNA isolated with the Maxwell method was significantly higher compared with the QIAamp method (P < 0.0001). Integrity of ccfDNA was significantly higher in the QIAamp isolate (P < 0.0001). Recovered mtDNA was not significantly different between both extraction methods used.ConclusionsThe choice of extraction method can significantly influence the ccfDNA output and integrity. Both methods, however, enabled isolation of sufficient ccfDNA for further downstream applications. With this approach, isolation of ccfDNA could enable the non-invasive detection and analysis of somatic mutation within endometriosis tissue.

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Condition tags

endometriosis

MeSH descriptors

Cell-Free Nucleic Acids Cell-Free Nucleic Acids Endometriosis Real-Time Polymerase Chain Reaction Adult Cell-Free Nucleic Acids DNA, Mitochondrial DNA, Mitochondrial DNA, Mitochondrial Endometriosis Female Fluorometry Fluorometry Humans Prospective Studies Real-Time Polymerase Chain Reaction

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