352_DNA quantification using absorbance (A260) and fluorescent methods (Qubit™ and Quant-iT™/PicoGreen™)

Andrea Krumm, BMG LABTECH GmbH, 77799 Ortenberg, Germany, 10/2020
  • BMG LABTECH multi-mode plate readers are compatible with common DNA quantification methods
  • Quantification of double-stranded DNA with hundreds of samples in parallel
  • Detect down to 0.8 pg double-stranded DNA per well

Introduction

Double-stranded DNA not only holds all information to build a human but is also a popular molecule to study. Whether expression levels or infections that are measured by PCR, or next generation sequencing to generate sequence information and to detect mutations responsible for disease. DNA-based methods require the quantification of DNA: here we present commonly used methods based on absorbance or fluorescence to quantify DNA at all concentrations, volumes, and in any throughput.

Materials & Methods

  • 384 well UV-transparent (Greiner #781801)
  • black 384 well small volume (Greiner #784076)
  • LVis Plate (BMG LABTECH)
  • CLARIOstar® Plus, VANTAstar®, FLUOstar® Omega (BMG LABTECH)
  • Qubit™ dsDNA BR (ThermoFisher #Q32850)
  • Qubit™ dsDNA HS (ThermoFisher #Q32854)
  • Quant-iT™ Picogreen™ (ThermoFisher #P11469)
  • Lambda DNA (ThermoFisher #SD0011)
  • Roti® Stock 100x TE and H2O (Carl Roth)

Experimental procedure 
Lambda DNA was diluted in 1x TE buffer to concentrations ranging between 1 pg/ml and 130 µg/ml. Qubit BR and Qubit HS assays were performed by diluting DNA 1:10 in the working solution. For 96 well plates, 20 µl DNA were mixed with 180 µl of dye solution, for 384 well plates, 2 µl of DNA were mixed with 18 µl of dye solution. In the Quant-iT/PicoGreen assay, DNA and working solution were mixed 1:1. A 96 plate well was filled with 100 µl of dye and 100 µl of DNA, a 384 well with 10 µl of each solution. Absorbance-based measurements employ the DNA as it is and volumes of 100 µl, 50 µl, and 2 µl were placed into a 96 well, 384 well, or LVis plate, respectively.

All measurements were done in triplicates and 12 replicates for blank measurements.

Instrument settings

 

Absorbance (predefined protocol dsDNA)
Optic settings

Absorbance, endpoint

Spectrum 1nm resolution 

220-360 nm

General settings

Number of flashes 22

Settling time 0.2 s (384)
Settling time 0.5 s (96)

 

 

Fluorescence intensity, CLARIOstar Plus and VANTAstar
Optic settings

Fluorescence intensity, endpoint, top

Monochromator

FITC default
Excitation 483-14
Auto dichroic
Emission 530-30

General settings

Number of flashes 20

Settling time 0.2 s

Start settings

Focus: Autofocus
Dynamic Range: EDR

 

 

Fluorescence intensity, FLUOstar Omega
Optic settings

Fluorescence intensity, endpoint, top

Filters

Excitation: 485
Emission: 520

General settings

Number of flashes 20

Settling time 0.2 s

Start settings

Gain adjustment On highest standard, target value 90%

Results & Discussion

Absorbance-based dsDNA quantification BMG LABTECH multi-mode microplate readers acquire absorbance spectra using an ultrafast UV/vis spectrometer. DNA spectra can be measured in volumes from 2-100 µl using different plate formats. The resulting absorbance spectra were automatically analyzed with the MARS analysis software. Next to DNA concentration, it calculates the A260/A280 ratio to determine protein contaminations (Fig 1).

Fig. 1: DNA quantification by UV/vis absorbance in a 384 well plate. Spectra were acquired between 220-360 nm on a BMG LABTECH microplate reader.

Fig. 2: Automatic analysis performed by BMG LABTECHs MARS software shows DNA concentrations (bold) and A260/A280 ratio.Fig. 3: A260 DNA quantification using an ultrafast UV/vis spectrometer and different plate formats. LVis plate was used with 2 µl, 384 well plate with 50 µl and 96 well plate with 100 µl. OD is plotted against DNA amount per well.

Fluorescence-based DNA quantification
Fluorescent dyes for the quantification of DNA are used because, in contrast to absorption, they are more specific and detect lower DNA concentrations. Here, we compared three commonly used dyes: Qubit high sensitivity and low sensitivity kits and the Quant-iT PicoGreen dsDNA quantification kit. All kits were measured according to the instructions of use and with the same standards.

Upon detection, the data were evaluated with the MARS analysis software. It calculated the concentration of unknowns, measured in parallel to the standard curve. The results and concentration ranges of the three assays are compared in Figure 3. It shows the Quant-iT and  Qubit HS kits covering a large concentration range and being sensitive for low DNA concentrations. The Qubit BR kit covers high DNA concentrations.

Fig. 4: Double-stranded DNA quantified with fluorescent assays and a CLARIOstar Plus. A 384 well plate was used with a final volume of 20 µl.

Comparison of plate formats and reader types
Further, the limit of detection was calculated for all three assay kits and the absorbance method. The detection limit is the concentration at the blank value plus three times the standard deviation of 12 blanks (mean blanks + 3*SDblank). The results are shown in Table 1 and figure 4. The maximal measurable concentrations for fluorescent dyes are taken from the kits specifications.
Fig. 5: Comparison of dsDNA quantification methods analyzed on the VANTAstar, FLUOstar Omega and CLARIOstar Plus. Lowest detection limit was calculated based on measurements shown here. Upper detection limit was taken from kit specification (fluorescent dyes) and reader specification (absorbance).
Table 1. Comparison of microplate-based DNA quantification methods regarding sample volume and minimum DNA input (for CLARIOstar Plus)

 

Method

Plate

Sample Volume

Minimum DNA input

A260 LVis 2 µl 5000 pg

A260

96 well

100 µl

33000 pg

A260

384 well

50 µl

10500 pg

QubitHS

96 well

1-20 µl

3.4 pg

QubitHS

384 well sv

0.1-2 µl

0.8 pg

Qubit BR

96 well

1-20 µl 

70 pg

Qubit BR

384 well sv

0.1-2 µl 13.6 pg

Quant-iT/ PicoGreen

96 well

100 µl

3.5 pg

Quant-iT/ PicoGreen

384 well sv

10 µl

2.3 pg

Conclusion

BMG LABTECH microplate readers reliably detect double-stranded DNA. The instruments cover quantification of any sample concentration and volume. The data shown above help to find the right instrument and quantification method for your needs.

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