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Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Author(s): Streleckiene G, Forster M, Inciuraite R, Lukosevicius R, Skieceviciene J

Publication: Biopreserv Biobank, 2019, Vol. , Page

PubMed ID: 31343271 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared extraction and quantification methods and investigated the effects of freeze-thaw cycling plasma and hemolysis on the measured quantity and size distribution of cfDNA fragments, gDNA contamination, and the yield of miR-223. To determine the effects of freeze-thaw cycling of plasma, blood was obtained from 1 healthy male and 4 healthy female donors into K₂EDTA tubes on two different days. Immediately after draw, blood was centrifuged at 2000 x g for 10 min with a smooth brake profile. Plasma was then pooled, aliquoted, and either immediately recentrifuged at 3000 x g for 10 min or freeze-thaw cycled to -20˚C and completely thawed at room temperature once or twice before centrifugation. To investigate the effects of hemolysis, three tubes of K₂EDTA blood were collected from one healthy volunteer on two different days. Moderate hemolysis was induced in one tube by drawing the specimen through a needle once, severe hemolysis was induced in another by drawing through a needle five times, and the third tube was processed without inducing hemolysis. Plasma was obtained as described above. Hemolysis was measured spectrophotometrically. Plasma was centrifuged a third time at 16000 x g for 10 min before cfDNA extraction. cfDNA was extracted using the NextPrep-Mag cfDNA Isolation Kit, the MagMAX Cell-Free DNA Isolation Kit, or the QIAamp Circulating Nucleic Acid Kit. cfDNA was analyzed immediately or after storage at 4˚C. cfDNA was quantified using Qubit dsDNA HS Assay and by laser-induced fluorescence-based microcapillary electrophoresis. miRNA-223 was quantified using the TaqMan miRNA Assay. Significance of differences between quantification methods was evaluated using the Mann-Whitney U-test and reported as ρ. Significance of freeze-thaw cycling and hemolysis was evaluated using the T-test (P-value).

   Summary of Findings:

   cfDNA quantification was method-dependent with higher mean levels reported using Qubit than capillary electrophoresis (7.11±3.24 ng/mL versus 4.06±1.55 ng/mL, ρ=1.86 x 10^-21) which the authors attribute to the lack of specificity of the Qubit method. The quantity of cfDNA reported by Qubit was significantly higher when extracted with the QIAamp Kit than when extracted with the NextPrep-Mag and MagMAX kits (ρ=1.62 x10^-7 and ρ=1.16x10^-6, respectively), but there were no differences in reported yield among the kits when quantified by capillary electrophoresis. There were 3 peaks of <1000 bp visible on the electropherograms (172-177 bp, 351-355 bp, and 511-583 bp), which the authors considered mono-, di-, and tri- nucleosomal cfDNA. The distribution of the three fragments depended on the extraction kit used with QIAamp yielding fewer mononucleosomal fragments and more dinucleosomal and trinucloeosomal fragments than NextPrep (ρ=0.0003, ρ=0.002, and not significant, respectively) and MagMAX kits (ρ=8.22 x 10^-5, ρ=0.002, and ρ=0.008 respectively).

   Freeze-thaw cycling of the plasma once and twice increased the amount of cfDNA isolated using the QIAamp Kit (P=0.016 and P=0.031, respectively) but did not impact yield of cfDNA using the MagMAX and NextPrep-Mag kits. A significant increase in the amount of mononucleosomal fragments after 1 freeze-thaw cycle was observed when extraction was with QIAamp (P=0.044) and after two cycles when extraction was with the MagMAX Kit (P=0.023). A significant increase in trinucleosomal fragments was observed after two freeze-thaw cycles when extraction was with the QIAamp Kit (P=0.044).  Further, there was no effect of freeze-thaw cycling on the yield of gDNA, regardless of extraction method. cfDNA yield was not affected by hemolysis, regardless of cfDNA extraction method, but there was significantly more gDNA obtained from the severely hemolyzed specimen than the non-hemolyzed specimen when extraction was with the MagMAX Kit (P=0.002).

   The amount of gDNA was higher when extraction was with the QIAamp and MagMAX kits than the NextPrep-Mag Kit (ρ=0.001 and ρ=0.004, respectively). The yield of miR-223 was higher using the QIAamp and NextPrep kits than the MagMAX Kit (ρ=0.003 and ρ=0.027, respectively).

    

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen
   Biospecimen Aliquots and Components	Hemolysis	Fine needle aspiration-induced Not induced
   Analyte Extraction and Purification	Analyte isolation method	NextPrep-Mag cfDNA Isolation Kit MagMAX Cell-Free DNA Isolation kit QIAamp Circulating Nucleic Acid Kit
   Fluorometry Specific	Technology platform	Automated capillary electrophoresis
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles

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