Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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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

Conclusion of Paper

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

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Melanoma
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Fluorometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(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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