NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays.

Author(s): Fernando MR, Jiang C, Krzyzanowski GD, Ryan WL

Publication: Clin Chim Acta, 2018, Vol. 483, Page 39-47

PubMed ID: 29655637 PubMed Review Paper? No

Purpose of Paper

This paper compared the fragment sizes of cell-free DNA in plasma, exosomes, and in the plasma pellet from whole blood collected from healthy non-pregnant and pregnant patients.

Conclusion of Paper

The number of amplifiable copies of beta actin in plasma and plasma exosomes from healthy non-pregnant individuals decreased with increasing amplicon length; indicating a small mean fragment size. In pregnant patients, the percentage cfDNA of ≥490 bp was higher both in plasma and plasma exosomes than in non-pregnant patients. Analysis of the pellet produced by centrifugation of plasma at 16,000 x g showed that the majority of the DNA was greater 1000 bp with smaller amounts of 600-1000 bp DNA present, but in pregnant patients there were also fragments of 150-200 bp that were absent in non-pregnant patients.

Studies

  1. Study Purpose

    This study compared the fragment size of cell-free DNA in plasma, exosomes, and in the plasma pellet from whole blood collected from healthy non-pregnant and pregnant patients. K2EDTA blood was collected from healthy volunteers and from women in the first trimester of pregnancy. Plasma was obtained by centrifugation at 1600 x g for 15 min at room temperature followed by centrifugation of the resultant plasma at 16,000 x g for 15 min. Exosomes were isolated from the plasma using the Invitrogen Total Exosome Isolation kit. cfDNA was extracted from plasma and exosomes were isolated using the QIAamp circulating nucleic acid kit. cfDNA was quantified by digital PCR amplification of four different length amplicons of beta-actin. DNA fragment size was also determined using a bioanalyzer.

    Summary of Findings:

    The number of amplifiable copies of beta actin in plasma and plasma exosomes from healthy non-pregnant individuals decreased with increasing amplicon length; indicating a small mean fragment size. Using the number of copies of the 76 bp product as baseline, only 39%, 18%, and 5.6% as many copies of the 135 bp, 490 bp, and 905 bp products were amplified in plasma from healthy individuals. Bioanalyzer analysis confirmed that the majority of the plasma cfDNA fragments were 150-200 bp, with a much smaller peak in the 300-500 bp range. Similarly, only 40%, 18%, and 3.3% as many of the 135 bp, 490 bp, and 905 bp products were amplified as the 76 bp fragment in exosomes from healthy individuals and analysis using a bioanalyzer showed the majority of fragments were 150-500 bp.

    In pregnant patients, the percentage cfDNA of ≥490 bp was higher both in plasma and plasma exosomes than in non-pregnant patients with visible peaks observed in the 300-500 bp and 1000-10,000 bp ranges. The relative percentage of the 135, 490, and 905 bp products compared to the 76 bp product was 34%, 14%, and 23% in plasma and 30%, 12%, 18% in exosomes.

    Bioanalyzer analysis of the pellet produced by centrifugation of plasma from healthy non-pregnant patients at 16,000 x g showed that the majority of the DNA was greater than 1000 bp with smaller peaks at 600-1000 bp Results were confirmed by dPCR with 100%, 87%, and 83% amplification of the 135 bp, 490 bp, and 905 bp products, respectively, relative to the 76 bp fragment. Bioanalyzer analysis of the pellet produced by centrifugation of plasma from pregnant patients also showed the majority of fragments were >1000 bp with a smaller number of fragments between 600-1000 bp, but there was also a DNA fragment of 150 and 200 bp detected, unlike the pellet from healthy patients.  dPCR confirmed the presence of some smaller DNA fragment with amplification 87%, 75%, and 68% amplification ofthe 135 bp, 490 bp, and 905 bp products, respectively relative to the 76 bp fragment.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Pregnant
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Digital PCR
    DNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition Pregnant
    Non-pregnant
    Biospecimen Aliquots and Components Blood and blood products Plasma exosomes
    Plasma
    Platelets
    Digital PCR Specific Length of gene fragment 76 bp
    135 bp
    490 bp
    905 bp
    Digital PCR Specific Targeted nucleic acid 76 bp
    135 bp
    490 bp
    905 bp
    Beta -actin
    Digital PCR Specific Technology platform Bioanalyzer

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