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, 11 (3), e0150528
eCollection

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

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

Yuki Nakayama et al. PLoS One.

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Dilution curves of Frozen-DNA diluted with distilled water as determined by NanoDrop, Qubit and qPCR.
Each Frozen-DNA sample was serially diluted with distilled water, and the concentration of each diluent was measured by NanoDrop (circles), BR-Qubit (squares), HS-Qubit (diamonds) and qPCR (triangles). The broken line shows the expected NanoDrop value. The concentration (ng/μl) of each original DNA solution measured by NanoDrop is shown at the top right: dilution ratio = 1. Two additional concentrations are also shown in each graph. The detection limits of NanoDrop, BR-Qubit, HS-Qubit and qPCR are 2 ng/μl, 2 ng/μl, 0.2 ng/μl and 1 pg/μl, respectively.
Fig 2
Fig 2. Electrophoresis of DNAs by Agilent 2200 TapeStation.
DNAs (100 ng/lane) were electrophoresed on an Agilent 2200 TapeStation. The DNA integrity number (DIN) indicates the fragmentation of genomic DNA on a scale from 1 to 10. A high DIN indicates highly intact DNA, and a low DIN indicates strongly degraded DNA. Lane 1, Frozen-H1; lane 2, Frozen-H2; lane 3, Frozen-H3; lane 4, FFPE-H1; lane 5, FFPE-H2; lane 6, FFPE-H3; lane 7, Trizol-h1; lane 8, Trizol-h2; lane 9, Trizol-h4; lane 10, Trizol-h6; and lane 11, Trizol-h7.
Fig 3
Fig 3. Quantification of Frozen-DNA diluted with various solutions by Qubit.
(A) Frozen-R2 DNA was serially diluted with distilled water (black) or TE buffer (white), and the concentration of each diluent was measured by BR-Qubit (square) and HS-Qubit (diamond). (B) Eleven Frozen-DNAs were diluted with distilled water or TE buffer to approximately 20 ng/μl, as measured by NanoDrop, and the concentration of each diluent was measured by HS-Qubit. The ratios of the Qubit to NanoDrop values were determined for each diluent. (C, D) Frozen-R1 DNA was serially diluted with distilled water (closed diamonds), 0.01 mM NaCl (open diamonds), 0.1 mM NaCl (squares), 1 mM NaCl (triangles) or 10 mM NaCl (circles). The broken line shows the expected NanoDrop values. The concentration (ng/μl) of each original DNA solution, as measured by NanoDrop, is shown at the top right: dilution ratio = 1. (D) The Q/E ratio was determined for each diluent, as shown in Fig 3C. (E) Frozen-R2 DNA was serially diluted with TE buffer (white) or distilled water (black), and a 0.1 volume of 100 mM Tris-HCl/10 mM EDTA was added to the latter diluent (gray). The expected NanoDrop values in parentheses indicate those diluted with distilled water. The detection limits of each measurement are described in Fig 1.
Fig 4
Fig 4. Quantification and qualification of FFPE-DNA.
(A) Each FFPE-DNA was serially diluted with distilled water, and the concentration of each diluent was measured by NanoDrop (circles), BR-Qubit (squares), HS-Qubit (diamonds) and qPCR (triangles). The broken line indicates the expected NanoDrop value. The concentration (ng/μl) of each original DNA solution, as measured by NanoDrop, is shown at the top right: dilution ratio = 1. The detection limits of each measurement are described in Fig 1. (B) Various lengths of the target sequence were amplified from Frozen- and FFPE-DNAs. The amplified products were electrophoresed on agarose gels. Lane 1, Frozen-H1; lane 2, Frozen-H2; lane 3, Frozen-H3; lane 4, FFPE-H1; lane 5, FFPE-H2; and lane 6, FFPE-H3.
Fig 5
Fig 5. Quantification and qualification of Trizol-DNA.
(A) Trizol-DNA was serially diluted with distilled water, and the concentration of each diluent was measured by NanoDrop (circles), BR-Qubit (squares), HS-Qubit (diamonds) and qPCR (triangles). The broken line indicates the expected NanoDrop value. The concentration (ng/μl) of each original DNA solution, as measured by NanoDrop, is shown at the top right: dilution ratio = 1. The detection limits of each measurement are described in Fig 1. (B) Various lengths of the target sequence were amplified from Trizol-DNAs, and the amplified products were electrophoresed on an agarose gel. Lane 1, Frozen-H1; lane 2, Trizol-h1; lane 3, Trizol-h2; lane 4, Trizol-h3; lane 5, Trizol-h4; lane 6, Trizol-h5; lane 7, Trizol-h6; and lane 8, Trizol-h7.
Fig 6
Fig 6. Quantification of ssDNA by ssDNA-Qubit and dsDNA-Qubit.
Frozen-R1 and Trizol-h3 diluted with TE buffer or distilled water in 20 ng/μl were measured with ssDNA-Qubit and dsDNA-Qubit. The amounts of dsDNA (black) and ssDNA (white) were determined using the standard curve shown in S1 Fig.

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Grant support

This work was supported in part by Grant-in-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.
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