Technical note A comparison of DNA quantification values obtained by UV spectrophotometry (including NanoDrop) and PicoGreen analysis Introduction to the DNA samples, signals are read and After completing DNA extraction from a biological concentrations determined using a standard curve. sample, it is common practice to quantify the DNA As PicoGreen® only binds to double-stranded prior to using it in downstream applications. To DNA, it has the advantage that the reported ensure optimal performance, reactions that utilise DNA concentration is an accurate estimation of nucleic acids typically specify the input quantities the quantity of DNA that is present within in the of DNA that they require. sample and is not influenced by the presence of ssDNA or RNA. A disadvantage of this method is There are a range of methods available for the that it cannot be used to provide an estimate of quantification of DNA including absorbance, DNA purity. agarose gel electrophoresis and fluorescent DNA- binding dyes. The traditional method involves DNA extracted in LGC’s service laboratories measurement of the absorbance of the sample is routinely analysed using traditional UV using a UV spectrophotometer. DNA has a spectrophotometry as this provides an estimation maximal absorbance near 260 nm so UV light of of both DNA quality and quantity. If required, DNA this wavelength is passed through the samples. can be quantified using PicoGreen® in addition to Higher levels of absorbance are indicative of spectrophotometry (subject to additional charge). greater concentrations of DNA present within the This study provides a comparison of estimated samples. This method has the advantage that concentration values obtained using three the quality of the DNA can also be assessed; quantification methods, and offers insight into the absorbance at 280 nm is also measured to repeatability of the three methods. determine the level of protein contamination. Method The A260 / A280 ratio is indicative of the purity of the DNA samples and values of 1.8 or higher A set of 44 DNA samples, extracted in LGC’s relate to pure DNA samples. A disadvantage of laboratory from human blood samples using this method is that single-stranded DNA (ssDNA) our Kleargene chemistry, were quantified using and RNA also absorb UV light at 260 nm and three different methods. Two different UV can therefore interfere with the results and cause spectrophotometry instruments were used – a overestimation of the double-stranded DNA traditional plate reader, the FLUOstar Omega, (dsDNA) concentration. A wide range of UV and the NanoDrop – and the samples were also spectrophotometers are available, varying from quantified using PicoGreen®. traditional instruments that quantify DNA in plates 1. UV spectrophotometry: FLUOstar Omega plate or cuvettes, to instruments such as the NanoDrop reader – DNA samples were arrayed into a 96- (Thermo Scientific) that are designed to quantify well plate. Absorbance was measured at 260 DNA from micro-volumes of sample. nm and 280 nm on an FLUOstar Omega plate PicoGreen® is a fluorescent nucleic acid stain that reader. The absorbance reads were performed selectively binds to dsDNA. It has an excitation three times. An advantage of this method is maximum at 480 nm and emission of fluorescence that the volume of DNA used for quantification can be read at 520 nm. PicoGreen® is added can be returned to the original sample post-analysis. 2. UV spectrophotometry: NanoDrop (Thermo these samples, to ensure that DNA was at an Scientific) – DNA samples were quantified appropriate concentration for the PicoGreen® individually using the NanoDrop. 1 µL of each standard curve. Diluted DNA was arrayed sample was dispensed onto the pedestal and in triplicate into a 384-well plate alongside absorbance measured. The NanoDrop software DNA standard curve samples. An appropriate calculates a DNA concentration value based on volume of PicoGreen® was dispensed into the absorbance values obtained. Each sample each well, and the plate incubated at room was quantified on the NanoDrop three times. temperature, protected from light, for 5 minutes. Fluorescent signal was measured at 520 nm on 3. PicoGreen – a DNA dilution was performed, a FLUOstar Omega plate reader. based on UV spectrophotometry results for Results and Discussion Figure 2 illustrates the difference in mean concentration obtained by the three quantification DNA concentration and purity methods for a subset of the 44 DNA Measured DNA concentration of the samples samples tested. differed significantly between the three methods of quantification (F2 = 15, P <0.0001). Reported 180 DNA concentration was highest when samples 160 were measured using the NanoDrop (mean = 134 140 ng / µL), and lowest when samples were measured 120 using PicoGreen (mean = 87 ng / µL). Figure 1 100 UV Spec: illustrates the mean measured DNA concentration NanoDrop 80 for the 44 DNA samples, obtained using three concentration (ng / µL) UV Spec: A 60 different quantification methods. FLUOstar Omega 40 Mean DN PicoGreen 140 20 130 0 DNA sample 120 Figure 2. Mean DNA concentration for a subset of 15 of 110 the 44 DNA samples analysed. Green, orange and blue 100 bars illustrate the concentration estimate from NanoDrop, concentration (ng / µL) A 90 FLUOstar Omega plate reader and PicoGreen® methods respectively. DNA concentrations are consistently estimated 80 as higher using the Nanodrop platform than the FLUOstar Mean DN 70 Omega or PicoGreen methods. UV Spec: NanoDrop UV Spec: FLUOstar PicoGreen Omega As PicoGreen® is specific to dsDNA, the lower Figure 1. Mean measured DNA concentration for the 44 concentration values determined using this DNA samples analysed. method will be more reliable estimations of actual dsDNA whilst the values obtained by both UV Individual paired t-tests were performed to spectrophotometry methods are likely to have been ascertain which measurement methods significantly influenced by presence of ssDNA and / or RNA. differed from each other; adjusted P-values of P <0.0001 were obtained for all comparisons, The mean A260 / A280 ratio obtained using the indicating that DNA concentrations obtained Omega FLUOstar data for the 44 DNA samples with one method differed significantly from those was 1.8 which is indicative of pure DNA, and was obtained using the other two methods. very consistent across all samples (mean standard deviation = 0.02). These values are similar to the values obtained using the NanoDrop (mean A260 / A280 ratio = 1.9, mean standard deviation = 0.02). Repeatability of measurements To determine the repeatability of PicoGreen® analysis in our laboratories, a second dilution As each individual DNA sample was quantified plate was prepared from the same original by each of the three methods in triplicate, the DNA samples and the measurement procedure repeatability of each method can be calculated. repeated. DNA concentrations calculated for For all three datasets, the standard deviation this second dilution plate were compared to (SD) for each sample was calculated using those from the original dilution plate. Strong the three individual concentration values. correlation (R2 = 0.98) was observed between The mean SD, calculated using SD values the two datasets indicating that calculated from all samples, was then calculated. The DNA concentrations from two dilutions were mean standard deviation values for the three very similar. datasets are very comparable, as detailed in Table 1, indicating that repeatability of the two methods is very similar. The maximum sample SD (highest SD for an individual sample) is Summary comparable between the FLUOstar Omega and The NanoDrop, the FLUOstar Omega and PicoGreen datasets, but is much higher for the PicoGreen® are all reproducible methods for the NanoDrop dataset. The minimum sample SD quantification of DNA samples, although the results values (lowest SD for an individual sample) are obtained using the NanoDrop were more variable, comparable between the three datasets. as indicated by the higher maximum sample SD value. DNA concentration values determined by UV spectrophotometry were significantly higher Standard Quantification Method than those determined by PicoGreen®. Of the deviation UV Spec: UV Spec: PicoGreen® two UV spectrophotometry-based instruments, (SD) ng/ NanoDrop FLUOstar the values obtained using the NanoDrop were µL Omega consistently higher than those obtained using plate the FLUOstar Omega. The consistently higher reader values for the UV spectrophotometry methods are Mean attributable to the inability of UV spectrophotometry sample 9 9 11 to distinguish between double-stranded and single- SD stranded nucleic acids. Maximum sample 67 24 26 Although quantification values from PicoGreen® SD analysis are a more reliable measure to use Minimum in calculations of how much dsDNA to use in sample 1 1 3 downstream applications, UV spectrophotometry SD analysis is important as it enables the purity of Table 1. Comparison of repeatability of three methods the DNA to be estimated. LGC perform traditional for DNA quantification, based on 44 DNA samples. UV spectrophotometry (FLUOstar Omega plate Standard deviation values are comparable between the reader) as a standard procedure on DNA extracted two methods. in our service laboratories, but offers PicoGreen® analysis as an additional service, should this be required. Key words: DNA quantification; spectrophotometry, NanoDrop, PicoGreen www.lgcgroup.com/genomics • [email protected] Science for a safer world Brazil