Using NanoQuant Plate™ for Fluorescence-Based DNA Quantification in Small Volume Samples

Introduction

Accurate DNA quantification is needed in several procedures of molecular biology. These procedures include:

  • Fragment purification
  • cDNA synthesis for the production of libraries
  • Quantitation of amplification products

Using absorbance measurements at 260 nm (A260) is the traditional way of determining the concentrations of nucleic acid samples. However, this method does not discriminate between RNA and DNA and is restricted in terms of sensitivity. An alternative strategy is to use fluorescent dyes that bind to DNA molecules. Using these dyes, nucleic samples can be quantified with a higher sensitivity.

The fully equipped spectrophotometer, Infinite® 200 NanoQuant can carry out absorbance measurements in microplates, and by using the specifically designed NanoQuant Plate, can accurately and easily determine small-volume nucleic acid samples.

Additionally, fluorescent samples as small as 2 μl can be the analyzed, because the Infinite® 200 NanoQuant can be upgraded with fluorescence-reading capabilities according to user’s convenience [1]. The Infinite® 200-series of instruments can perform measurements with the NanoQuant Plate.

This article describes the use of the Infinite® 200 multimode reader and the related NanoQuant Plate for fluorescence-based DNA quantification in small quantities of samples using an ultra-sensitive fluorescent dye called Pico Green. This dye is used for the quantitation of double-stranded DNA (dsDNA).

Material and methods

Instrument and plates

  • Infinite® M200 Quad4 monochromator detection system (Tecan, Austria)
  • 96-well flat bottom polystyrol microplates (Greiner BioOne, Germany)
  • NanoQuant Plate with 16 individual sample positions with integrated quartz optics (Tecan, Austria)

Reagents and additional materials

  • Quant-iT PicoGreen quantitation reagent (Invitrogen, CA, USA)
  • 20x Tris/EDTA buffer (Sigma)
  • Lambda DNA standard, 100 μg/ml (Invitrogen, CA, USA)
  • ddH2O
  • laboratory kimwipes
  • 70% ethanol
  • single- and multi-channel pipettes with suitable tips

Reagent preparation

To prepare the reagent, Tris/EDTA (TE) buffer was diluted from 20x to 1x in ddH2O. Both the Lambda DNA standard and the Quant-iT PicoGreen reagent were diluted using 1x TE.

According to the manufacturer’s instructions, the Quant-iT PicoGreen quantitation reagent was freshly prepared as a 200-fold dilution of the stock solution. From every dilution, 50 μl was transferred to a 96-well microplate and combined with 50μl of the PicoGreen solution to achieve a final volume of 100 μl. A blank containing 50 μl TE buffer with PicoGreen quantitation reagent was also included.

DNA dilution series

Table 1 shows the summary of the dilution of the Lambda DNA standard from 1000 to 0.9 ng/ml using 1x TE buffer.

Table 1. Dilution series of standard Lambda DNA in TE buffer

Sample

DNA concentration

[ng/ml]

DNA concentration per 2 μl (NQP)

1

1000

2.0 ng

2

500

1.0 ng

3

250

0.5 ng

4

125

0.25 ng

5

62.5

0.12 ng

6

31.3

0.06 ng

7

15.6

0.03 ng

8

7.8

0.015 ng

9

3.9

0.007 ng

10

1.9

0.003 ng

11

12

0.9

0.002 ng

0 (blank)

0

 

Instrument settings

The NanoQuant Plate needs to be used in standard i-control mode for fluorescence measurements.

Using high-pressure compressed air and an ultrasonic bath, the NanoQuant Plate was cleaned according to the corresponding Quick Guide instructions, before making all measurements [1]. Table 2 lists the parameters according to which a measurement script was set up in i-control V1.5.

Table 2. Measurement parameters and instrument settings on Infinite M200 NanoQuant with Tecan i-control software

Measurement settings on Infinite® M200 NanoQuant

Parameter

Setting

Plate

Tecan 16 Flat Black [NanoQuant Plate]

Part of the plate

A1-H2

Fluorescence

Top reading mode

Excitation

485 nm

Bandwidth

9 nm

Emission

535 nm

Bandwidth

20 nm

Gain

optimal

Flashes

25

Integration time

20 μs

 

2 μl from each DNA dilution mixed in the ratio of 1:1 with the PicoGreen quantitation reagent was transferred from the microplate well onto the samples spots of the NanoQuant Plate. The plate’s lid was then closed carefully. Table 2 summarizes the script with which the plate was measured. At least three independent sample replicates were used to perform all measurements.

Results

Linearity

Using Microsoft Excel, standard deviations (stdev), average values, and coefficient of variations (CVs) were calculated after all raw values were blank-corrected. Slope and coefficient of determination (R2) were measured after plotting the average values in a line diagram and adding a trend line.

NQP measurements of Lambda DNA dilutions ranging from 1000 to 0.9 ng/ml demonstrate good linearity values, leading to an R2 value of 0.9966, as shown in Figure 1.

Figure 1. Linearity of fluorescence signals from DNA dilutions

Sensitivity

Table 3. Average RFU and stdev of triplicate DNA dilutions from 1000 to 0.9 ng/ml stained with PicoGreen

DNA concentration

[ng/ml]

Average RFU

stdev

CV [%]

1000

44285

40.31

0.09

500

20821

353.55

1.70

250

12691

41.72

0.33

125

7260

376.89

5.19

62.5

3636

323.15

8.89

31.3

2935

114.55

3.90

15.6

2615

169.00

6.46

7.8

2194

66.47

3.03

3.9

2247

21.92

0.98

1.9

1437

152.74

10.63

0.9

1479

224.15

15.15

0 (blank)

1281

132.94

10.38

 

The 250 ng/ml sample was chosen as a representative DNA concentration, and the signal of this sample was used to calculate the sensitivity of PicoGreen-based DNA quantification. The formula given below was used to calculate the detection limit:

8.7 ng/ml dsDNA is the calculated detection limit which corresponds to about 17 pg dsDNA in a 2 μl sample volume as it is employed in the NanoQuant Plate.

Table 4. Detection limit of PicoGreen-based DNA quantification using the NanoQuant Plate

Sample

Detection limit per ml

Detection limit per 2 μl (NQP)

250 ng/ml

8.7 ng

17 pg

 

Discussion

The basis for different types of applications in molecular biology is the accurate detection and sensitive quantification of nucleic acids in samples.

A reliable and sensitive technique for the quantification of small dsDNA amounts down to 17 pg is afforded by the Quant-iT PicoGreen system in tandem with the small volume measurement properties of the NanoQuant Plate.

This value of 17 pg is much lower than the quoted detection limit of the Quant-iT PicoGreen kit which is specified to be 25 pg in a 200 μl assay volume. It may be noted that the detection range of the Quant-iT PicoGreen system is improved, making it even more sensitive than specified, thanks to the capability of the NanoQuant Plate to analyze small volumes of samples (2 μl).

Conclusion

The combination of Infinite® 200 and the NanoQuant Plate provides a reliable and time-saving method for fluorescence-based quantification of dsDNA. The method enables obtaining highly accurate and sensitive results by consuming minimum amount of sample.

Abbreviations

  • cDNA                complementary DNA
  • CV                    coefficient of variation
  • dsDNA              double-stranded DNA
  • NQP                 NanoQuant Plate
  • RFU                 relative fluorescence unit(s)
  • RNA                 ribonucleic acid
  • ssDNA              single-stranded DNA
  • stdev                standard deviation
  • TE                    Tris/EDTA

Literature

  1. Quick Guide NanoQuant Plate, 2008 (Tecan Austria)
  2. Singer VL et al.: Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Analytical Biochemistry, 249, 228 - 238 (1997)

About Tecan

Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Jun 27, 2017 at 6:15 AM

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