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

The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

Author(s): Ponti G, Maccaferri M, Manfredini M, Kaleci S, Mandrioli M, Pellacani G, Ozben T, Depenni R, Bianchi G, Pirola GM, Tomasi A

Publication: Clin Chim Acta, 2018, Vol. 479, Page 14-19

PubMed ID: 29309771 PubMed Review Paper? No

Purpose of Paper

This paper investigated the effects of diagnosis on the concentration of cell-free DNA (cfDNA) in plasma specimens and compared differences observed between quantification methods.

Conclusion of Paper

Cell-free DNA concentrations were higher in patients with malignant melanoma than in patients with prostate cancer or healthy individuals. Using the Qubit ss-DNA, cfDNA levels were found to be higher than when quantified by any of the other methods and using real-time PCR (qPCR) resulted in the lowest levels. Bland-Altman analysis revealed that the differences in quantification between qPCR and any other method was less than one standard deviation. 

Studies

  1. Study Purpose

    This study investigated the effects of diagnosis on the concentration of cfDNA in plasma specimens and compared differences observed between quantification methods. Plasma was obtained from EDTA blood from 18 patients with malignant melanoma, 67 patients with prostate cancer, and 15 healthy individuals by centrifugation at 4˚C at 1600 x g for 10 min then at 16000 x g for 10 min. Plasma was aliquoted and stored at -80˚C until DNA extraction. DNA was extracted using the QIAamp circulating nucleic acid kit and stored at -20˚C. DNA was quantified using the Qubit dsDNA HS Assay Kit, the Qubit ssDNA Assay Kit, spectrophotometry, and real-time PCR amplification of a 67 bp region of APP.

     

    Summary of Findings:

    Cell-free DNA concentrations were higher in patients with malignant melanoma than in patients with prostate cancer or healthy individuals. Using the Qubit ss-DNA, cfDNA levels were found to be higher than when quantified by any of the other methods and using qPCR resulted in the lowest levels. Bland-Altman analysis revealed that the differences in quantification between qPCR and any other method was less than one standard deviation. Quantified levels of cell-free DNA were strongly correlated between Qubit ssDNA and qPCR (R=0.766) and modestly correlated between Qubit dsDNA and qPCR (R=0.689), NanoDrop and Qubit ds-DNA (R= 0.618), NanoDrop and Qubit ssDNA values (R=0.599), and Nano-Drop and qPCR (R=0.442).

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    • Neoplastic - Melanoma
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    DNA Spectrophotometry
    DNA Fluorometry
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition Malignant melanoma
    Prostate cancer
    Healthy
    Fluorometry Specific Technology platform Qubit ss-DNA
    Qubit ds-DNA
    Spectrophotometry
    Real-time PCR
    Real-time qPCR Specific Technology platform Spectrophotometry
    Qubit ds-DNA
    Qubit ss-DNA

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