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Isolation and Quantification of Plasma Cell-Free DNA Using Different Manual and Automated Methods.

Author(s): Polatoglou E, Mayer Z, Ungerer V, Bronkhorst AJ, Holdenrieder S

Publication: Diagnostics (Basel), 2022, Vol. 12, Page

PubMed ID: 36292239 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the yield and fragment size profile of cfDNA isolated from plasma of healthy volunteers using six different methods. Blood was collected from ten healthy volunteers into K3EDTA blood tubes and stored at room temperature for <60 min before plasma separation. Plasma was separated by centrifugation at 1,600 g for 10 min followed by 6,000 g for 10 min and aliquots were stored at -80°C. Plasma was thawed at room temperature and cfDNA was isolated using the following commercial kits: QIAamp Circulating Nucleic Acid Kit, NucleoSpin Plasma XS Kit, QIAmp MinElute ccfDNA Mini Kit, Cf-Pure Cell-Free DNA Extraction Kit, MagMAX Cell-Free DNA Isolation Kit, and the automated MagNA Pure 24 Total NA Isolation Kit. cfDNA was stored in LoBind tubes at -20°C. cfDNA was quantified using the Qubit dsDNA HS Assay and fragment size profiles were evaluated using the High-Sensitivity DNA Kit with a Bioanalyzer.

   Summary of Findings:

   cfDNA was successfully isolated from plasma using each of the six extraction kits evaluated, but cfDNA yield (determined by fluorometry) was significantly higher when extraction was with the QIAamp Circulating Nucleic Acid Kit than the NucleoSpin Plasma XS(P=0.0001), QIAmp MinElute ccfDNA Mini Kit (P<0.0001), or MagMAX Cell-Free DNA Isolation Kit (P=0.0004); while cfDNA yield was lower using the NucleoSpin Plasma XS Kit compared to  either the QIAamp MinElute ccfDNA Mini Kit or MagMAX Cell-Free DNA Isolation Kit. However, cfDNA yields were comparable when extraction was with the QIAmp Circulating Nucleic Acid Kit, cf-Pure Cell-Free DNA Extraction Kit, and MagNA Pure 24 Total NA Isolation Kit. Interestingly, cfDNA yields were lower when determined by bioanalyzer than fluorometry. When cfDNA yield was assessed by bioanalyzer, NucleoSpin Plasma XS and cf-Pure Cell-Free DNA Extraction Kits yielded significantly less DNA than either the QIAamp MinElute ccfDNA Mini Kit (P=0.0052 and P=0.0335, respectively) or the MagMAX Cell-Free DNA Isolation Kit (P=0.03496 and P=0.0377, respectively). The smallest CV in yield was observed when the QIAamp Circulating Nucleic Acid Kit (11.8%) or the MagNA Pure 24 Total NA Isolation Kit (16.0%) were used. All kits showed a bias toward recovery of 50-250 bp DNA.  The percentage of cfDNA represented by mono-nucleosomes varied among the isolation kits evaluated but differences were nonsignificant (QIAmp MinElute ccfDNA Mini Kit: 34.5%; MagMAX Cell-Free DNA Isolation Kit: 34%; QIAamp Circulating Nucleic Acid Kit: 26.5%; cf-Pure Cell-Free DNA Extraction Kit: 22%; MagNA Pure 24 Total NA Isolation Kit: 22%; and NucleoSpin Plasma XS: 21%).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Circulating Nucleic Acid Kit NucleoSpin Plasma XS Kit QIAmp MinElute ccfDNA Mini Kit Cf-Pure Cell-Free DNA Extraction Kit MagMAX Cell-Free DNA Isolation Kit Automated MagNA Pure 24 Total NA Isolation Kit
   Automated electrophoresis/Bioanalyzer Specific	Technology platform	Bioanalyzer Qubit

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