Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions

RESEARCH ARTICLE Pitfalls of DNA Quantification Using DNABinding Fluorescent Dyes and Suggested Solutions Yuki Nakayama, Hiromi Yamaguchi, Naoki Einaga, Mariko Esumi* Department of Pathology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan * Abstract OPEN ACCESS Citation: Nakayama Y, Yamaguchi H, Einaga N, Esumi M (2016) Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions. PLoS ONE 11(3): e0150528. doi:10.1371/ journal.pone.0150528 Editor: Hodaka Fujii, Osaka University, JAPAN Received: August 25, 2015 Accepted: February 15, 2016 Published: March 3, 2016 Copyright: © 2016 Nakayama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported in part by Grantin-Aid for Scientific Research (C) 25430142 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/) received by ME, and Nihon University Multidisciplinary Research Grant (M14-012) from Nihon University (http://www.nihon-u.ac.jp/) received by ME. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR. Introduction Recently, various simple quantification methods have been developed to determine DNA concentrations in trace amounts of samples. These techniques have been useful in medicine, including in molecular diagnosis and prognosis, e.g., the detection of cell-free fetal DNA in PLOS ONE | DOI:10.1371/journal.pone.0150528 March 3, 2016 1 / 12 Pitfalls of DNA Quantification by DNA-Binding Dyes Competing Interests: The authors have declared that no competing interests exist. maternal circulation and circulating tumor cells [1–5]. Recently, clinical samples have also been subjected to novel technologies, such as next-generation sequencing (NGS) in translational research. It has become important to more accurately evaluate the quality and quantity of DNA. The following three methods are used to quantify DNA: 1) UV absorbance measurement at 260 nm (UV spectroscopy); 2) fluorescence measurement of a fluorescent dye, such as PicoGreen, specifically bound to double-stranded DNA (dsDNA) (fluorescence spectroscopy) [6]; and 3) relative quantification of a particular DNA sequence based on real-time PCR (quantitative PCR; qPCR) [7]. The UV spectroscopy method measures the maximal absorbance of nucleic acids; thus, it does not distinguish between dsDNA, single-stranded DNA (ssDNA), RNA and nucleotides. In contrast, the fluorescence spectroscopy method determines the amount of intact dsDNA, and the quantitative value yielded decreases with the level of fragmentation and denaturation of DNA [8–11]. Therefore, fluorescence spectroscopy using PicoGreen is more useful than UV spectroscopy for evaluating the template activity of DNA for PCR. In this regard, qPCR accurately quantifies the amount of the target sequence and is the ideal method for checking the quantity of DNA used for NGS [12]. However, qPCR takes much more time and is more expensive than fluorometry or UV spectroscopy. Simbolo M et al. have suggested that the ideal workflow for quantifying DNA, especially DNA extracted from histopathological tissues suitable for NGS, is first to assess the presence of contaminants in the sample with a UV spectrometer (NanoDrop) and subsequently to use a fluorescence spectrometer (Qubit) to quantify dsDNA [13]. However, it is unknown whether Qubit can be completely replaced by qPCR for determination of DNA concentrations. In the present study, we quantified the following three types of DNA using the three quantification methods NanoDrop, Qubit and qPCR: DNA extracted from fresh frozen tissues, formalin-fixed paraffin-embedded (FFPE) DNA and DNA extracted from the remaining fraction after RNA extraction with Trizol reagent (Life Technologies). We found inconsistencies in DNA quantification between Qubit and qPCR and proposed the optimum combinations of the aforementioned DNA quantification methods. Materials and Methods Samples Fresh frozen non-tumorous liver tissues were obtained from six patients with liver metastasis of colorectal carcinoma by surgical resection and from five Long Evans Cinnamon rats (Frozen-H1 to H6 and R1 to R5). The freshly frozen livers were stored at -80°C until use. FFPE samples were prepared from the same human liver tissues as described above (FFPE-H1 to H3) with fixation in 10% buffered formalin for 2 to 4 days. Human non-tumorous liver tissues were also obtained from the resection of seven cases with HCV-positive hepatocellular carcinoma (Trizol-h1 to h7), and RNA was extracted using Trizol reagent (Life Technologies, Waltha (...truncated)