Quantification of circulating cell-free DNA by fluorimetry (Qubit) and spectrophotometry (NanoDrop) in patients with malignant melanoma and prostate cancer

Abstract

6 Department of Diagnostic and clinical medicine and public health, University of Modena and Reggio Emilia, Modena Background: Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. Several studies highlighted the role of cfDNA levels in the prognostic assessment of different malignancies. The quantification of pure cfDNA is a prerequisite for a reliable genotyping focused on cancer-specific DNA mutations. In the present study, the quality and quantity of the cfDNA were assessed by three different quantification procedures, Qubit with single (ss) and double strand (ds) DNA assay kit and NanoDrop. The aim of the study was to identify the accuracy and the potential applications of these techniques in preliminary cfDNA quantification. Materials and methods: We examined blood samples of 13 patients affected by melanoma, 10 patients affected by prostate cancer and 5 healthy controls. Blood samples were collected at the diagnosis of advanced malignant melanoma or prostate cancer or at the time of enrollment for the healthy controls. Samples were processed within an hour from plasma collection; cfDNA was extracted from plasma through Qiagen kit and Promega automatic extractor. Qubit 2.0, with ss-and dsDNA assay kit, and NanoDrop 1000 were used to measure the total amount of cfDNA. Results: The quantification by Nanodrop (mean value 13.57 ng/µl), Qubit ssDNA (mean value 32.80 ng/µl) and dsDNA (mean value 5.42 ng/µl) assay kits revealed differences among the three procedures, highlighting that fluorimetric quantification with ssDNA probe offers a more sensitive and accurate method than spectrophometric measurements. In particular, Qubit ssDNA probe revealed higher sensitivity in the quantification of small amounts of pure single and dsDNA. Our data demonstrated a clear correlation between cfDNA concentrations and tumor stage. Conclusions: The spectrophotometric NanoDrop approach showed better quality and purity assessment of extracted cfDNA and could be used for the cfDNA preliminary qualification of plasma samples. The sequential combination of both methods should be adopted for a cost-effective preliminary assessment of total cfDNA in melanoma and prostate cancer patients. In addition, cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers.