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Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.

Author(s): Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, Huggett JF

Publication: Anal Bioanal Chem, 2014, Vol. 406, Page 6499-512

PubMed ID: 24853859 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare ccfDNA yields using four different extraction kits and to determine the effects of different PCR-based quantification methods, and plasma and elution volumes. Purchased plasma from 17 donors was stored at -80°C until use. Specimens were centrifuged at 5000 rpm, and specimens 1-5 and 6-17 were pooled. Plasma pools were aliquoted for storage at -80°C and fragmented ADH plasmid was added to half of the pools. DNA was extracted using the QIAamp CNA kit, NucleoSpin Plasma XS kit, FitAmp plasma/serum DNA isolation kit and the QIAamp DNA blood mini kit.

   Summary of Findings:

   Extraction with the QIAamp CNA kit generated ccfDNA yields that were 2.2-2.5 fold higher than when DNA was extracted using the QIAamp DNA blood mini kit, while <50% as much DNA was obtained using the NucleoSpin kit, and DNA yields using the FitAmp plasma/serum DNA isolation kit were at or below the limit of detection. Extraction with the QIAamp CNA kit allowed for >80% recovery of 115, 461 and 1448 bp fragments of ADH (albeit with slightly reduced recovery of the 115 bp fragment compared to the 461 bp fragment, 83% versus 99%, p<0.05), while recovery of ADH fragments following extraction with the DBM kit was highly size dependent with only 21% recovery of the 115 bp fragment and 37% recovery of the 461 bp fragment, and 58% recovery of the 1448 bp fragment(p<0.001 and p<0.05, respectively). The CT values for probe-based real-time PCR were not affected by the volume ratio of plasma to eluate, but SYBR green CT values were significantly higher when the volume ratio of plasma to elutant was 250 than when it was ≤125 using the CNA kit (p<0.0001). No effect was observed for the plasma volume to elution volume ratios examined by either real-time PCR assay when DNA was extracted with the NucleoSpin kit, but the kit did not allow an input per µL extract of>60. ccfDNA yield obtained with the CNA kit increased linearly with plasma input up until 3 mL, after which yield plateaued. Quantification of ccfDNA levels by dPCR rather than by qPCR resulted in the same rank order of copies/mL of tTERT, RPPH1 and ERV3, but the magnitude of the differences was greater with qPCR and a greater than 2-fold difference were observed for levels of seven reference genes. Reliability was improved when three reference genes were used to determine ccfDNA yield.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	0.6 mL 1.0 mL 2.0 mL 3.0 mL 5.0 mL
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	5-20µL
   Real-time qPCR Specific	Targeted nucleic acid	ADH ALUJ TERT ERV3 GAPDH NAGK RPH1 VP
   Real-time qPCR Specific	Detection method	Probe-based Sybr-based
   Real-time qPCR Specific	Length of gene fragment	115 bp 461 bp 1448 bp
   Analyte Extraction and Purification	Analyte isolation method	QIAamp CNA kit NucleoSpin Plasma XS kit FitAmp plasma/serum DNA isolation kit QIAamp DNA blood mini kit
   Real-time qPCR Specific	Technology platform	dPCR

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