Abstract
BackgroundNon-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®).ResultsStatistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient.ConclusionsThis comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.
Highlights
Non-invasive molecular analysis of cell-free DNA became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA in noninvasive prenatal test
The cell-free DNA (cfDNA) concentrations measured by QUBIT HS were statistically different than those measured by the two other DNA quantification methods (QUBIT HS vs. digital droplet PCR (ddPCR), p = 0.01; QUBIT HS vs. BIABooster, p = 0.01; ddPCR vs. BIABooster, p = 0.78)
When cfDNA concentration was measured by QUBIT HS, the highest cfDNA amount were obtained using the IDEAL
Summary
Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In cancerous tissues, the size of cfDNA varies a lot, because in addition to apoptosis, necrosis and autophagy are responsible of the death of cancer cells. In this case, the fragments can reach 450–500 bp [3]. An innovative capillary electrophoresis system, called BIABooster based on μLAS technology, allows simultaneous DNA concentration and separation operations With this system, the sensitivity of cfDNA detection reach 20 pg/mL [7]. A range of commercially kits are today available for cfDNA extraction from plasma, which might influence quantification of cfDNA, the fragment size distribution of cfDNA, and the chimerism detection, i.e. donor cfDNA quantification, under different pathological conditions and cfDNA quantification and qualification methods used
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