Extraction of Cell-Free DNA: Evaluation of Efficiency, Quantity, and Quality.
Author(s): Terp SK, Pedersen IS, Stoico MP
Publication: J Mol Diagn, 2024, Vol. , Page
PubMed ID: 38336350 PubMed Review Paper? No
Purpose of Paper
The purpose of this study was to compare the yield, fragment size, percentage of double-stranded DNA, and day-to-day variability of DNA extracted from the plasma of healthy volunteers using four different methods.
Conclusion of Paper
Recovery of the spike-in control and yield of the two targeted endogenous cfDNAs were significantly higher when extraction was with the manual or automated (QIAcube) QIAamp Circulating Nucleic Acid Kit than with the QIAamp MinElute ccfDNA Kit with a QIAcube instrument or the QIAsymphony DSP Circulating DNA Kit with a QIAsymphony instrument. All methods yielded an acceptable ratio of long to short amplicons (<0.4, indicating low lymphocyte contamination) and more than 50% of isolated fragments were less than 700 bp, but some differences were observed between methods. The extraction methods evaluated did not differ in the percentage of isolated DNA that was double-stranded. The between-day variance in spike-in DNA recovery or levels of either endogenous cfDNA were comparable among the extraction kits evaluated.
Studies
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Study Purpose
This study compared the yield, fragment size, percentage of double-stranded DNA, and day-to-day variability of DNA extracted from the plasma of healthy volunteers using four different methods. Blood was collected from eighteen healthy volunteers into four EDTA tubes on two different days. Plasma was obtained by dual centrifugation at 2000 g for 10 minutes at 4°C within 2 h of blood collection. The four plasma specimens from the same venipuncture were pooled and spiked with a 191 bp fragment cysteine-rich polycomb-like protein (CPP1) mRNA (5800 copies/mL) before aliquoting. Plasma aliquots were stored at -80°C until cfDNA extraction. cfDNA was extracted from 1 mL aliquots of plasma using the Manual QIAamp Circulating Nucleic Acid Kit, the QIAamp Circulating Nucleic Acid Kit with a QIAcube instrument, the QIAamp MinElute ccfDNA Kit with a QIAcube instrument, or the QIAsymphony DSP Circulating DNA Kit with a QIAsymphony instrument. Extracted cfDNA was stored in LoBind or Qiagen collection tubes at -20°C until analysis. Levels of ribonuclease P/MRP subunit P30 (RPP30) and ER membrane protein complex subunit 7 (EMC7) mRNA and the spike-in CPP1 were quantified by droplet digital PCR (ddPCR). cfDNA fragment size was analyzed using a TapeStation instrument and cfDNA was defined as 50-700 bp. Contamination with high molecular weight DNA was analyzed by amplification of long (250 bp) and short (65 bp) amplicons of EMC7 with a ratio <0.4 considered to be low contamination and >0.7 as high contamination.
Summary of Findings:
Recovery of the spike-in control was comparable when extraction was with the manual and automated (QIAcube) QIAamp Circulating Nucleic Acid Kit but was significantly higher than either the QIAamp MinElute ccfDNA Kit with a QIAcube instrument (P<0.001, both) or the QIAsymphony DSP Circulating DNA Kit with a QIAsymphony instrument (P<0.001, both). Similarly, the number of copies of recovered RPP30 was significantly higher when extraction was with the manual or automated QIAamp Circulating Nucleic Acid Kit than with the QIAamp MinElute ccfDNA Kit with a QIAcube instrument (P=0.009 and P=0.0011, respectively), or the QIAsymphony DSP Circulating DNA Kit with a QIAsymphony instrument (P=0.013 and P=0.0016, respectively). Levels of EMC7 were higher when extraction was with the manual or automated QIAamp Circulating Nucleic Acid Kit than with the QIAamp MinElute ccfDNA Kit with a QIAcube instrument (P=0.001, both), or the QIAsymphony DSP Circulating DNA Kit with a QIAsymphony instrument (P=0.001, both). Levels of RPP30 and EMC7 were strongly correlated, regardless of extraction method (R=0.988). Significantly fewer acceptable droplets/per well were found when extraction was with the QIAamp MinElute ccfDNA Kit with a QIAcube instrument than the QIAamp Circulating Nucleic Acid or QIAsymphony Kits (P<0.001, both). The ratio of long to short amplicons, a marker of lymphocyte DNA contamination, was less than the specified threshold of 0.4 in all extractions; however, the ratio was significantly higher when extraction was with the QIAsymphony DSP Circulating DNA Kit with a QIAsymphony instrument than extraction with the other methods evaluated (P<0.0001). The percentage of isolated DNA fragments that were between 50 and 700 bp (corresponding to cfDNA) was >50% in all extractions and for all extraction methods evaluated; the average percentage of DNA fragments that were cfDNA-sized was >75%. However, extraction with the QIAamp MinElute ccfDNA Kit using a QIAcube instrument resulted in a higher percentage of cfDNA-sized fragments than when extraction was with any of the other methods evaluated (P=0.0001). The percentage of isolated DNA that was double-stranded did not differ among the methods tested. The between-day variance in spike-in DNA recovery or levels of either endogenous cfDNA were comparable among the extraction kits investigated.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Digital PCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Manual QIAamp Circulating Nucleic Acid Kit
QIAamp Circulating Nucleic Acid Kit with QIAcube
QIAamp MinElute ccfDNA Kit with QIAcube
QIAsymphony DSP Circulating DNA Kit with QIAsymphony
Biospecimen Acquisition Time of biospecimen collection Two different days compared
Digital PCR Specific Targeted nucleic acid Spike-in CPP1
RPP30
EMC7
Digital PCR Specific Length of gene fragment 65 bp
250 bp