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Fluorometric Quantitation of dsDNA using PicoGreen下載
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January 25, 2017
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Using an Agilent BioTek Synergy Neo2 multimode reader to quantify DNA in solution
Author: Paul Held, PhD, Agilent Technologies, Inc.
An essential element of cellular and molecular biology is the ability to quantitate DNA in large numbers of samples at a sensitivity that enables determination of small amounts of sample. This application note describes a method to quantify dsDNA using an Agilent BioTek Synergy Neo2 multimode reader.
Many techniques of cellular and molecular biology require the ability to quantify dsDNA in large numbers of samples at sensitivities that only require a small amount of the total sample. Isolation of plasmids from bacterial cultures, genomic DNA from mammalian cells, cDNA synthesis for library production, and quantitation of PCR products for diagnostic purposes all require the direct quantitation of dsDNA. Also, many biochemical studies that involve the growth kinetics of cell cultures or cell cycle studies require normalization by DNA content.
The most commonly used method for the determination of nucleic acid concentration is the determination of absorbance at 260 nm (A260) as described by Matiatis et al.1 This method, while quite adequate for many situations, can suffer from the interfering absorbance of contaminating molecules. Many of these contaminates which include nucleotides, RNA, EDTA, and phenol are commonly found in nucleic acid preparations. As a result, several fluorescent staining techniques have been developed to alleviate many of the problems associated with absorbance at 260 nm.2-4 One such stain is PicoGreen, which, in conjunction with the Agilent BioTek Synergy Neo2 multimode reader, offers high specificity as well as high sensitivity for dsDNA quantitation.
Materials and methods
Quant-iT PicoGreen Reagent (part number P11495), Salmon Sperm DNA (part number 15632011), and TE (100x) buffer concentrate (part number BP-1338-1) were purchased from Thermo Fisher Scientific. Solid 96-well black microplates, (part number 3915), were from Corning. Herring sperm DNA was diluted to 2 µg/mL with TE (10 mM Tris, 1 mM EDTA, pH 7.5) as the diluent. The final concentration was confirmed using 260 nm absorbance. A series of dilutions ranging from 0.0 to 2,000 ng/mL of purified Herring Sperm was made using TE and 100 µL aliquots pipetted into microplate wells. Equal amounts (100 µL) of working PicoGreen quantitation reagent were mixed and incubated for 10 minutes at room temperature, protected from light. Working PicoGreen reagent was prepared by diluting the concentrated DMSO‑PicoGreen stock solution, provided in the PicoGreen kit, 1:200 with TE according to the kit instructions.2 Following incubation, the fluorescence was determined using an Agilent BioTek Synergy Neo2 multimode reader. With filter‑based measurements, the reader used a 485 nm, 20 nm bandwidth excitation filter and a 528 nm, 25 nm bandwidth emission filter along with a 510 nm cutoff dichroic mirror. Monochromator-based determinations were made with an excitation wavelength of 485 nm and an emission wavelength of 528 nm, with the reader default bandwidth. The reader was programmed to Autoscale the sensitivity setting to the highest DNA concentration. The data were collected from the top using a 0 msec second delay after plate movement, 25 reads per well. Emission spectral scans were made by fixing the excitation at 485 nm (9 nm bandwidth) and varying the emission wavelengths in 1 nm increments. Excitation scans were produced by fixing the emission wavelength at 528 nm and varying the excitation wavelengths in 1 nm increments. All bandwidths were set to 9 nm and the PMT gain setting set at 125 using normal read speed. Reactions were typically determined within one hour.
Results and discussion
Fluorescent spectral scans of dsDNA (1 µg/mL) stained with PicoGreen reagent demonstrate excitation and emission peaks at 485 nm and 528 nm respectively after blank subtraction (Figure 1). These wavelengths were then used for subsequent measurements in order to maximize fluorescence signal for DNA titration experiments.
Figure 1. Fluorescent excitation and emission spectrum of dsDNA (1 µg/mL) stained with PicoGreen reagent. Data were normalized such that maximum value was 10,000 RFUs.
The fluorescence intensity was determined for DNA concentrations ranging from 0 to 1,000 ng/mL. Over this range the intensity increased in a linear fashion. Using Agilent BioTek Gen5 data reduction software, a least means squared linear regression analysis can be generated with a coefficient of determination (R2) value of 0.998. The average coefficient of variance (%CV) of the standards was less than 3%, with the greatest variation in the lower DNA concentrations tested (data not shown). In terms of sensitivity, the assay was found to be sensitive to the picogram level. Under appropriate sensitivity settings, DNA concentrations as low as 30 pg/mL were found to be statistically different (P <0.05) from the TE only, 0 ng/mL, control. Quantitation of dsDNA using the fluorescent properties of PicoGreen, in conjunction with the Agilent BioTek Synergy Neo2, allows researchers to quantitate as little as 6 pg/well (30 pg/mL in a 0.2 mL total volume), thus providing reliable quantitation of dsDNA concentrations ranging over four orders of magnitude.
Figure 2. Linearity of spectral filter-based detection. Fluorescence of dsDNA titration incubated with PicoGreen reagent was made using a 485/528 Agilent BioTek Synergy Neo filter cube. Concentration ranged from 0.0 to 1 µg/mL of dsDNA with least means squared linear regression analysis. Insert figure depicts the data points for the lower concentrations (0 to 1,000 pg/mL). Data points represent the mean values of eight determinations at each concentration.
In addition to filter cubes for fluorescence detection, the Synergy Neo2 can be configured with dual monochromators for both excitation and emission. Similar linearity can be observed using the monochromators for wavelength selection as compared to the filter-based detection (Figure 3). Similar detection limits (30 pg/mL) were observed with the monochromators as with the filter-based optical system.
Figure 3. Linearity of monochromator-based detection. Fluorescence of dsDNA titration incubated with PicoGreen reagent was made using an Agilent BioTek Synergy Neo 2 monochromator. Excitation was set to 485 nm and the emission was set to 528 nm, with a bandwidth of 20 nm for both excitation and emission. Concentration ranged from 0.0 to 1 µg/mL of dsDNA with least means squared linear regression analysis. The inset figure depicts the data points for the lower concentrations (0 to 1,000 pg/mL). Data points represent the mean values of eight determinations at each concentration.
Despite the lower magnitude in raw scale, the monochromators provide a better signal to background ratio as compared to the filters, as demonstrated in Figure 4. This is primarily the result of lower background measurements produced by the dual monochromators.
Figure 4. . Comparison of signal to background ratios of monochromatorand filter-based optical paths. The ratio of each sample to the mean of the blank (0 µg/mL dsDNA) was plotted. Data represent the mean and standard deviation of eight determinations.
The Agilent BioTek Synergy Neo2 multimode reader is a highly configurable multimode microplate reader. This study examined the filter-based and monochromator-based fluorescence detection optical systems. Filter-based detection is provided with an array of predefined filter cubes that have barcode identification. This allows the reader to ensure that the correct filter combination is used for the intended fluorescent moiety. The spectral characteristics of the filters and dichroic mirrors employed maximize the excitatory light on target as well as the recovery of the emitted signal, maximizing sensitivity. The dual monochromator system, while resulting in lower raw signal than the filter system, offers complete flexibility in regards to wavelength selection as well as bandwidth. In addition, the high-resolution design of the monochromators results in very low background fluorescence, which translates into better signal-to-blank (S/B) ratios than the filter‑based optics.
To achieve optimal sensitivity, several measures can be undertaken. Removal of certain common contaminates, such as proteins or organics (e.g. phenol and butanol), will result in tighter CVs and lower background signal, resulting in an increase in sensitivity. Several compounds are known to decrease the fluorescent signal when present in a PicoGreen‑DNA assay. In particular, the presence of 200 nM sodium chloride or 50 mM magnesium chloride can decrease the signal by approximately 1/3. In cases where only low DNA concentrations are to be determined, a calibration curve using lower concentrations can be used. Additionally, the use of microplates that use lower fluorescent plastics would be expected to reduce background, and therefore increase sensitivity.
- Maniatis T., E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
- PicoGreen dsDNA Quantitation Reagent and Kit instruction, (1996) Molecular Probes, Eugene, OR.
- Labarca, C. and K. Paigen (1980) A Simple rapid, and sensitive DNA assay procedure. Analytical Biochemistry 102:344-352.
- Ogura, M., C. Keller, K. Koo, M. Mitsuhashi (1994) Use of the Fluorescent Dye YOYO-1 to Quantify Oligonucleotides Immobilized on Plastic Plates. BioTechniques 16:1032-1033