Performance of Spectrophotometric and Fluorometric DNA Quantification Methods

Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric (i.e., a NanoDrop instrument) and three fluorometric (i.e., the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) methods. An analysis of variance (ANOVA) scheme was used to determine the influence of the analyst, the method, and the combination of analyst and method, on DNA quantification. For most samples, the measured DNA concentration was close to or slightly above the concentration of 10 ng/μL as specified by the supplier. Results obtained by the three analysts were equal. However, it was found that, compared to the fluorometric kits, the used spectrophotometric instrument in the case of fish DNA samples tends to overestimate the DNA concentration. Therefore, if sufficient sample volume is available, a combination of a spectrophotometric and a fluorometric method is recommended for obtaining data on the purity and the dsDNA concentration of a sample.


Introduction
Quantification of the exact amount of dsDNA in a sample is very important within a wide variety of molecular biology applications [1][2][3]. In order to avoid wasting samples in cases where only a limited amount of sample is available, which is often the case in, e.g., forensic and clinical settings, a reliable quantification method is required. Several methods are available on the market which can handle mass-limited samples, each with their own benefits and limitations.
The NanoDrop is a spectrophotometric instrument that measures the absorption of light at 260 nm to determine the amount of DNA in a sample. ssDNA, dsDNA, and RNA absorb at this wavelength, and therefore this method cannot discriminate between these types of nucleic acids. To obtain an indication of the purity of a sample, the 260/280 nm and the 260/230 nm ratios are determined. A ratio of 1.7-2.0 for 260/280 nm is acceptable (pure DNA has a ratio of 1.8), whereas a lower value can be caused by protein or phenol contamination. When RNA (or ssDNA) is present in a sample, this results in a higher ratio. For the 260/230 nm ratio, a value of more than 1.5 is indicative for a DNA sample of good quality. Since the NanoDrop determines the absorbed light at 260 nm, it tends to give higher values for the measured concentration than methods that are dsDNA-specific (e.g., Qubit) [1,3,4]. The expected accuracy and reproducibility are 2% and about 2 ng/µL for samples below 100 ng/µL, respectively [5].
Fluorometric methods are widely used for DNA quantification. These kits contain an intercalating dye, such as PicoGreen, that binds in between the DNA strands of dsDNA. The fluorescent signal that is measured is related to the DNA concentration. The Qubit fluorometer, in combination with the Qubit High Sensitivity quantification kit, can be used for sample concentrations of 10 pg/µL till 100 ng/µL [6]. The AccuGreen quantitation kit is a recently developed dsDNA quantification method that can be used with a fluorometer, such as the Qubit fluorometer. According to the manufacturer, this kit is suitable for samples in the range of 0.1-10 ng/µL [7]; however, detailed information about this kit is not yet available in the literature. These two kits, based on fluorescent intercalating dyes, cannot give an indication of the purity of a sample. The AccuClear Ultra High Sensitivity kit with seven standards contains a green fluorescent dye (468/507 nm) that is compatible with fluorescence microplate readers. Samples between 0.03 and 250 ng are within the linear range of this kit [8]. Additionally, for this kit, only data provided by the supplier are available and-similar to the other two investigated kits-this kit also cannot determine the purity of the DNA sample.
UV absorbance spectroscopy (e.g., with a NanoDrop instrument) has been compared previously to several other DNA quantification methods (e.g., Qubit, SYBR Green, and PicoGreen dye staining). Haque et al. concluded that spectrophotometric DNA quantification was the most concordant and precise method in comparison with the PicoGreen assay and a real-time quantitative genomic PCR assay [9]. Simbolo et al. showed, for two DNA samples with a known concentration, that the NanoDrop and Qubit overestimated and underestimated the DNA concentration, respectively [10]. Nielsen et al. encountered higher DNA concentrations than expected based on the manufacturers information with, among other methods, UV spectroscopy and SYBR Green dye staining [2]. Nakayama et al. compared the Qubit with the NanoDrop and qPCR and also concluded that the Qubit, depending on the method for DNA extraction and dilution (e.g., salt concentration and denatured DNA), tends to underestimate the amount of DNA [11]. Additionally, He et al. measured significantly lower concentrations of DNA with the broad-range Qubit assay compared to the absorbance value. The AccuGreen assay and the high-sensitivity Qubit assay gave concentrations that were comparable to the spectrophotometric measurements [12]. The level of fragmentation does not influence spectrophotometric measurements, but this method has the lowest sensitivity when compared to PicoGreen and qPCR. The accuracy of PicoGreen and qPCR is influenced by fragmented DNA according to Sedlackova et al. [13]. Hussing et al. compared various quantification methods, among which were spectrophotometry with a NanoDrop instrument and fluorometry with a Qubit system. For all samples tested, e.g., adapter-dimer-rich, fragmented, and PCR-inhibited libraries, the NanoDrop gave much higher concentration values compared to the Qubit measurements, of which the latter were comparable with the quantification results from electrophoresis-based methods [14]. Additionally, high-molecular-weight DNA is difficult to quantify due to the complexity of the sample [12]. Li et al. compared PicoGreen with the diphenylamine reaction method and UV absorbance and concluded that the latter is the best method for measuring impurities. PicoGreen performed best with degraded DNA samples, and in the case of contaminants, diphenylamine would be the method of choice [15]. The Qubit and the NanoDrop were used by Masago et al. to determine the RNA and DNA concentration of samples extracted from lung cancer patients. They concluded that the absolute DNA concentration determined with the NanoDrop was higher than that found with the QuBit. The concentration of RNA, however, showed no significant difference between the Qubit and the NanoDrop measurements [16]. Quantification is also important when analyzing circulating cell-free tumor DNA (cfDNA). Ponti et al. compared the NanoDrop and both the ssDNA and dsDNA kit for the Qubit with cfDNA samples. The ssDNA kit gave the highest average value, 23.08 ng/µL, while the NanoDrop and dsDNA kit gave average values of 8.48 ng/µL and 4.32 ng/µL, respectively. Additionally, qPCR was performed, which gave a much lower average value of only 0.39 ng/µL of cfDNA. Ponti et al. advised to use both the NanoDrop and the Qubit ssDNA kit in sequential combination in order to have a cost-effective solution for cfDNA quantification and only to use qPCR in the case of discordant values [17]. Khetan et al. found that for concentrations below 2.71 ng/µL, the NanoDrop was neither precise nor accurate. They recommend to use a fluorometric method for the quantification of cfDNA in blood samples, such as the Qubit [18]. In conclusion, the literature indicates that it can occur that spectrophotometric methods (slightly) overestimate the DNA concentration in comparison with fluorometric methods.
In this research, seven different DNA samples (four control samples from the tested kits and three in-house available DNA samples) and MilliQ ultrapure water as negative control were analyzed with one spectrophotometric method and three fluorometric methods. For the spectrophotometric analysis, a NanoDrop instrument was used, and for the fluorometric methods, the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA High Sensitivity Assay kit were used. The goal of this research is, besides comparing the quantification methods as was performed in the above described literature, to determine the influence of the factor analyst. Therefore, all samples and methods were tested by three analysts to determine the variance between persons.

Materials
The Qubit ® dsDNA HS Assay kit, including a 10 ng/µL standard DNA sample (Q) (λ dsDNA), was purchased from Thermo Fisher Scientific, Nieuwegein, NL, USA. The AccuClear Ultra High Sensitivity kit and the AccuGreen High Sensitivity kit (gift from Biotium), including 10 ng/µL standard DNA samples (AC and AG) (calf thymus dsDNA) in both kits, were obtained from Biotium. TaqMan ® Control Genomic DNA (TM) (human, male, 10 ng/µL) was purchased from Applied Biosystems ® . The TaqMan and AccuClear vials contained a limited amount of DNA, and to ensure that the same DNA sample was used in all the methods, a stock solution was made of 1 ng/µL TaqMan DNA (AccuGreen, AccuClear, and Qubit experiment) and AccuClear DNA (AccuGreen and Qubit experiment) prior to the experiments. All analysts used the same 1 ng/µL stock solution. Additionally, several other DNA samples were tested: 10 ng/µL salmon DNA (S) (D1626, Sigma-Aldrich, Zwijndrecht, NL, USA), 10 ng/µL herring DNA (H) (74782, Sigma-Aldrich), and 10 ng/µL DNA from Jurkat cells (J).

Spectrophotometric DNA Quantification Measurements with the NanoDrop Instrument
For the NanoDrop measurements, a Nanodrop 2000c instrument (Thermo Fisher Scientific) was used. MilliQ ultrapure water (M) was used as blank measurement, and absorption at 340 nm was used as baseline. After each measurement, the pedestals were wiped with a clean wipe (KIMTEX). A total of 1.5 µL of each DNA sample was measured in triplo by all three analysts.

Measurement with the AccuGreen High Sensitivity Kit
The protocol of the manufacturer was used for the measurements with the AccuGreen High Sensitivity kit. Each DNA sample was measured in triplo by all three analysts with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific).

Measurement with the AccuClear Ultra High Sensitivity Kit
The protocol of the manufacturer was used for the measurements with the AccuClear Ultra High Sensitivity kit. Each DNA sample was measured in triplo by all three analysts in a Corning 96 flat bottom black polystyrene microplate with a Tecan M200 PRO multimode reader, operated by Tecan I-control software.

Measurements with the Qubit dsDNA HS Assay Kit
The protocol of the manufacturer was used for the measurements with the Qubit dsDNA HS Assay kit. Each DNA sample was measured in triplo by all three analysts with a Qubit 2.0 Fluorometer.

Statistical Analysis
To determine whether there are significant differences per method, one-way ANOVA was conducted (α = 0.05). To check if the factors 'analyst', 'method', or 'analyst × method' were significant, ANOVA with repeated measures with two within-subjects factors was used, while taking sphericity into account. The Excel add-in "Real Statistics Using Excel" was used to carry out the ANOVA analyses (α = 0.05) [19]. The measured concentrations of the DNA samples are depicted in Figure 1 and can also be found in Table 1. Most of the DNA samples gave a value of 10 ng/µL ± 2 ng/µL, which is within the specifications of this method.   The measured concentrations of the DNA samples with the AccuGreen High Sensitivity kit are depicted in Figure 2 and can also be found in Table 1. The Qubit cannot measure values below 0.50 ng/mL, so this is displayed as 0 in Figure 2. This was the case for all the MilliQ ultrapure water (negative control) samples. Additionally, values above 600 ng/mL give a notification error ("fluorescence signal too high") which implies that no further quantification can be performed. This happened for two samples (once for the Qubit control and once for the AccuGreen control) of analyst 1, which means the original sample had a concentration above 12 ng/µL (these values were not included in the averaged data given in Figure 2).

Measurement with the AccuClear Ultra High Sensitivity Kit
The measured concentrations of the DNA samples with the AccuClear Ultra High Sensitivity kit are depicted in Figure 3 and can also be found in Table 1. Seven DNA standards are provided with the kit in order to generate a standard curve, by averaging the triplicate value for each sample. The equation of the trend line of this standard curve is used to calculate the amount of unknown DNA in each well. The AccuClear DNA sample was only quantified once by analyst 1, as can be seen in Table 1, due to a pipetting mistake. One well contained a double amount of DNA sample, and one well received no sample at all; a mistake that became clear from the fluorescence measurements.

Measurements with the Qubit dsDNA HS Assay Kit
The measured concentrations of the DNA samples with the Qubit dsDNA HS Assay kit are depicted in Figure 4 and can also be found in Table 1. Similarly to the AccuClear Ultra High Sensitivity kit, values below 0.50 ng/mL are displayed as 0 in Figure 4. This was the case for all the MilliQ ultrapure water (negative control) samples.

Statistical Analysis
By using one-way ANOVA, with the values given in Table 1, it turned out that the factor 'analyst' did not result in significant differences (sample concentrations) for each method (Tables A1-A4). To determine the influence of the factors 'method' and 'analyst × method', an ANOVA with repeated measures was performed. The two-factor ANOVA with repeated measures with two within-subjects factors showed that the factors 'analyst' and 'analyst × method' did not show a significant difference. In contrast, the factor 'method' did show a significant difference (p ≤ 0.05) ( Table A5). The differences in the mean values are also depicted in Figure 5. Upon comparison with the fluorometric DNA quantification methods, the spectrophotometric method using the NanoDrop instrument overestimated the DNA concentrations, as can be observed in Figure 5. This can be explained by the measured DNA concentration of the fish samples, which was, on average, 8.7 ng/µL for the spectrophotometric method and 0.8 ng/µL for the fluorometric methods.
To check if the variances of the differences between all factors are equal, sphericity must be determined. In the case that the variances of the differences between all combinations of related groups are equal, sphericity must be taken into account, which is the case when epsilon is equal to 1. The factor 'analyst' shows an epsilon of (close to) 1 for both the Greenhouse-Geisser (GG) and the Huynh-Feldt (HF) epsilon. However, the factors 'method' and 'analyst × method' show epsilon values far below 1 (Table A6). Using the corrected values, the factor 'method' is not significantly different (Table A7) [19].

Discussion
Measuring the DNA concentration of a sample with a spectrophotometer has several advantages. The method is fast, no additional reagents are required, no calibration is needed (besides measuring the blank), and the sample can be reused. Whereas with a standard spectrophotometer relatively large volumes are needed for cuvette measurements (in the order of milliliters), the NanoDrop instrument does not require cuvettes, and even volumes as low as 1 µL can be used. The main drawback of this spectrophotometric DNA quantification method is its nonspecificity: all compounds that absorb at 260 nm will contribute to a measurement, and no distinction between dsDNA, ssDNA, and RNA can be made. Apparently, the fish DNA samples contain a substantial amount of unknown specimen that is not dsDNA (according to the investigated fluorometric methods) that exhibits absorption at 260 nm.
Fluorometric methods used to measure the concentration are dsDNA-specific. These methods require more sample preparation steps, since the fluorescent dye (and additional buffer) must be added to each sample. It is also mandatory to create a standard curve with the fluorometer (as is the case for the AccuGreen High Sensitivity kit and the Qubit dsDNA HS Assay kit), or afterwards with software (e.g., Excel, as is the case for the AccuClear Ultra High Sensitivity kit). Preferentially, a new standard curve is made before each new set of measurements (Qubit readings) or per well plate (AccuClear). For Qubit readings, only two standards, 0 and 10 ng/µL, are available within the kit, which makes the standard curve a bit questionable. When the quality of one of the standards is compromised (e.g., contamination or pipette error), the curve is not trustworthy anymore, possibly going unnoticed by the analyst. The AccuClear Ultra High Sensitivity kit has seven standards included, measured in triplo, which makes this standard curve more reliable. The Qubit dsDNA HS Assay kit requires an incubation time of 2 min, and the AccuGreen High Sensitivity kit prescribes an incubation time of at least 2 min. Therefore, these latter fluorometric methods are relatively time-consuming in the case of a large amount of samples. With the AccuClear Ultra High Sensitivity kit, a whole 96-well plate (including reference samples) can be read at once. This makes this method more suitable for larger amounts of samples.
Although the fish samples, salmon and herring DNA, showed the best results in terms of purity, these samples did not contain 10 ng/µL according to the fluorometric methods. This is striking, since the sample does contain a DNA concentration of 10 ng/µL based on weighing (original sample is in solid state and must be diluted by the analyst to the appropriate concentration) and performed spectrophotometric measurements. With UV spectroscopy, all sources of nucleic acids, single-and double-stranded, are measured, while the fluorometric methods are dsDNA specific. Apparently, these fish samples do not contain the amount of dsDNA that is expected based on weight. Carvalho et al. used salmon sperm DNA samples as it turned out that the λDNA standard was not representative for fragmented DNA. The low-molecular-weight salmon sperm DNA is less purified and more fragmented. They measured dsDNA concentrations for the salmon sperm DNA, which were only around 10% of the expected concentration [20], which is in accordance with the results of this research. He et al. suggest to use a nucleic acid standard that matches the samples that are being measured [12].
For a spectrophotometric reading using the NanoDrop instrument, a sample volume of 1 µL is sufficient, while the fluorometric methods require 10 µL (AccuGreen and AccuClear) or 1-20 µL (Qubit). However, the fluorometric methods have a lower detection limit in comparison with methods based on absorbance, and therefore a more diluted sample can be used. The drawback of the fluorometric methods is that the purity, the 260/280 nm and 260/230 nm ratios, of the sample cannot be determined. Therefore, a combination of the NanoDrop (or another spectrophotometric method) in combination with a fluorometric method is recommended, in agreement with the suggestion of Simbolo et al. [10].
For all fluorometry-analyzed DNA samples, the expected concentration of 10 ng/µL was measured, since calibration standards of the the Qubit High Sensitivity quantification kit, the AccuGreen quantitation kit, and one of the standards of the AccuClear Ultra High Sensitivity kit contained this concentration. Additionally, this concentration falls within the range of measurable concentrations as indicated by the suppliers of the kits.
Some DNA quantification measurements showed pretty large standard deviations (≥0.5 ng/µL), so it is recommended to perform all measurements in triplo. Additionally, the used DNA quantification methods are nonspecific to the species. Moreover, it should be mentioned that, in contrast to commercial DNA samples, real-life/case samples (such as a buccal swab or a bone sample) might contain DNA from multiple biological sources. Therefore, in areas such as forensic genetics, human-specific DNA quantification methods are used (e.g., qPCR). In fact, it is noted that such real-life/case samples can (negatively) affect spectrometric as well as fluorometric DNA quantification.
Since epsilon is lower than 0.70 for the factor 'method', an MANOVA might be used, instead of an ANOVA. However, this is not recommended for this sample size (eight samples), which is lower than k (the number of levels of the repeated measures factor) + 10 [19,21].

Conclusions
A total of four different DNA quantification methods were investigated by three analysts for seven DNA samples and one blank. Based on the conducted ANOVA, it can be concluded that the factors 'analyst' and 'analyst × method' do not result in significant differences in sample concentration. In contrast, the factor 'method' does show a significant difference; in the case of fish samples, the applied spectrophotometric method overestimated the DNA concentration in comparison to the fluorometric methods used. This can be explained by the measured DNA concentration of the fish (herring and salmon DNA) samples, which was, on average, 8.7 ng/µL and 0.8 ng/µL for the spectrophotometric method and fluorometric methods, respectively. Presumably, these DNA samples contain a substantial amount of material that exhibits absorption at 260 nm, which is not dsDNA according to the fluorometric methods. Except for these fish samples, the measured samples show a concentration around 10 ng/µL, as is expected based on the information of the supplier. The fluorometric methods (the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) do not show a significant difference among the samples or analysts. To conclude, in order to achieve information on the purity and the dsDNA concentration of a sample, a combination of a spectrophotometric and a fluorometric method is recommended, provided that enough sample volume is available.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: