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

A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA.

Author(s): Lin SY, Luo Y, Marshall MM, Johnson BJ, Park SR, Wang Z, Su YH

Publication: Diagnostics (Basel), 2021, Vol. 11, Page

PubMed ID: 33916811 PubMed Review Paper? No

Purpose of Paper

This paper compared the yield and size profile of cell-free DNA (cfDNA) isolated from urine and plasma specimens of healthy donors using three different extraction kits with and without an additional clean-up step. The yield of fetal cfDNA isolated from plasma and urine specimens collected from pregnant women using three different extraction kits was also compared.

Conclusion of Paper

While all three kits were able to isolate mononucleosomal DNA from urine and plasma, the size profile of the extracted DNA from urine differed between the extraction methods evaluated in some urine specimens. Further, di- and tri-nucleosomal DNA was only observed in plasma from four patients when DNA was extracted with the JBS cfDNA Extraction Kit or the MagMAX Cell-Free DNA Extraction kit. The recovery of the mononucleosomal-size spike-in control from plasma and urine significantly improved when DNA was extracted with the MagMAX Cell-Free DNA Extraction Kit or the QIAamp Circulating Nucleic Acid Kit and was cleaned with the DNApure Cleanup Kit. Although levels of TP53 varied widely among donors, TP53 levels in plasma and urine were not affected by extraction method when the clean-up column was applied. Levels of fetal (y-chromosomal) cfDNA in urine were significantly higher when extraction was with the JBS cfDNA Extraction Kit than the QIAamp Circulating Nucleic Acid Kit, but levels of fetal DNA in plasma were comparable between the two methods (MagMAX Cell-Free DNA Extraction kit not compared).

Studies

  1. Study Purpose

    This study compared the yield, and size profile of cfDNA isolated from urine and plasma specimens of healthy donors using three different extraction kits with and without an additional clean-up step. The yield of fetal cfDNA isolated from plasma and urine specimens of pregnant women using three different extraction kits was also compared. All specimens from healthy donors were purchased from commercial sources or were archival-deidentified specimens from prior studies. No further details of procurement were provided. Frozen urine specimens from 3 healthy men and 3 healthy women were mixed with EDTA. Donor blood in EDTA tubes was purchased and plasma was obtained by centrifugation at 2800 g for 20 min, passed through 0.2 μm filter, and aliquoted.  For fetal DNA studies, urine was collected at two and three timepoints from 2 women carrying male fetuses, and blood was collected at 5 timepoints from 3 women carrying male fetuses, which included the women who contributed two urine specimens. Plasma was obtained from the blood of pregnant women by centrifugation at 3500 rpm and was subsequently stored at -20°C. Urine and plasma were spiked with a 141 bp fragment of DNA after which DNA was extracted from 3-4 aliquots of each plasma/urine specimen using the JBS cfDNA Extraction Kit, the MagMAX Cell-Free DNA Extraction kit, and the QIAamp Circulating Nucleic Acid Kit. Some aliquots of DNA that were isolated using the MagMAX Cell-Free DNA Extraction Kit and the QIAamp Circulating Nucleic Acid Kit were subjected to an additional purification step using a DNApure Cleanup Kit. Extracted DNA was frozen at -20°C. DNA was size-profiled using the High Sensitivity D5000 ScreenTape assay on the TapeStation 4200 instrument. TP53 and the Y-chromosome were amplified by real-time PCR. Protein contamination was measured using the Qubit Protein Assay Kit.

    Summary of Findings:

    While all three kits were able to isolate mononucleosomal DNA from the urine of 5 of the 6 healthy donors (three females and two males), the DNA yield in the urine specimen from the remaining donor (male) was extremely low. The size profile of the extracted DNA from urine differed among the three extraction methods for 2 of the 6 patients evaluated. Protein contamination was not detected in any of the DNA isolates. Recovery of the mononucleosomal-size spike-in control was superior when extraction was with the JBS cfDNA Extraction Kit than the MagMAX Cell-Free DNA Extraction kit or the QIAamp Circulating Nucleic Acid Kit (P<0.001). DNA recovery using the MagMAX Cell-Free DNA Extraction Kit or the QIAamp Circulating Nucleic Acid Kit improved significantly when a clean-up step was performed with the DNApure Cleanup Kit (P<0.001, both) to the extent that recovery was comparable to the JBS cfDNA Extraction Kit. Levels of TP53 varied widely between donors but were not affected by extraction method when the clean-up column was applied. Levels of fetal (y-chromosomal) DNA in urine were significantly higher when extraction was with the JBS cfDNA Extraction Kit than the QIAamp Circulating Nucleic Acid Kit (P<0.05).

    Similar to urine, mononucleosomal DNA was obtained from plasma specimens using all three of the DNA extraction kits evaluated, but while di- and tri-nucleosomal DNA was isolated from the plasma of four donors using the JBS cfDNA Extraction Kit and the MagMAX Cell-Free DNA Extraction kit, these peaks were not observed when extraction was performed with the QIAamp Circulating Nucleic Acid Kit. DNA recovery using the MagMAX Cell-Free DNA Extraction kit and the QIAamp Circulating Nucleic Acid Kit improved significantly when a clean-up step was performed with the DNApure Cleanup Kit (P=0.013 and P<0.001, respectively). Levels of TP53 in plasma were not affected by extraction method when the clean-up column was applied. Levels of fetal (y-chromosomal) DNA in plasma were comparable when extraction was with the JBS cfDNA Extraction Kit or the QIAamp Circulating Nucleic Acid Kit (the MagMAX Cell-Free DNA Extraction kit was not included in the comparison).

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    • Pregnant
    Platform:
    AnalyteTechnology Platform
    DNA Automated electrophoresis/Bioanalyzer
    Protein Fluorometry
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method JBS cfDNA extraction kit
    MagMAX Cell-Free DNA Extraction kit
    QIAamp Circulating Nucleic Acid Kit
    Real-time qPCR Specific Targeted nucleic acid TP53
    Y-chromosome
    Analyte Extraction and Purification Analyte purification No additional purification
    Purification with the DNApure cleanup kit

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