Using Spark® Multimode Microplate Reader and NanoQuant Plate™ for DNA Quantification and Purity Checks in Small Volumes

Introduction

Driven by the extensive use of high-throughput nucleic acid analysis approaches, including next-generation sequencing, reliable and fast quantification and purity checks of nucleic acids are becoming increasingly vital for many laboratories. To keep pace with the downstream processes, many techniques need accurate, rapid analysis of small sample volumes.

Fitted with patent-pending High Speed Monochromators (HSM), the new Spark® multimode microplate reader enables reproducible, accurate, and ultra-fast absorbance measurements.

Tecan’s Spark® microplate reader

An enhanced measurement range from 200 to 1,000 nm is provided by this unique optical system that ensures optimal performance across the entire range, especially for absorbance measurements in the deep UV range, including the purity checks for A260/A230 nucleic acids.

Using the patented NanoQuant plate, shown in Figure 1, the Spark® reader can also measure low volume samples in fluorescence and absorbance modes. By using just 2 μl sample volumes, this unique quartz optic enables simultaneous measurements of up to 16 samples.

Figure 1. Tecan’s NanoQuant Plate for low volume nucleic acid quantification.

The most popular method used to determine the concentration of nucleic acids is based on measuring the absorbance at 260 nm (A260). The absorbance values at 230, 260 and 280 nm (260/230 and 260/280 ratio ratio) can be compared to assess the purity of the RNA or DNA sample.

Sample contamination with phenols and proteins (aromatic groups) is indicated by a 260/280 ratio value of ≤2.0 for RNA or ≤1.8 for DNA, and contamination with salts, carbohydrates, or organic solvents is indicated by a 260/230 ratio ≤2.0.

For nucleic acid quantification, a preconfigured, ‘one-click’ application is offered by the SparkControl software which makes the process extremely fast and easy. The absorbance at 230, 260 and 280 nm is automatically measured by the reader.

The reader also performs a fast spectral scan from 200 to 1000 nm (in increments of 1 nm), often taking only five seconds per sample and preventing issues seen with sample evaporation. The SparkControl software is then used to display and analyze the resulting absorbance spectrum.

This article describes the utility of the Spark® reader for the quantification of low volume DNA, employing the NanoQuant Plateto measure 2 μl DNA samples – including a full spectral analysis – all in a fraction of seconds.

Materials and methods

  • Spark® multimode reader (Tecan, Austria)
  • NanoQuant Plate
  • Tris-EDTA (TE) buffer (BioThema, Sweden)
  • 96-well UV-Star®, flat bottom, transparent microplates (Greiner Bio-One, Austria)
  • Phage Lambda-DNA, 300 μg/ml (Invitrogen, USA)
  • ddH2O
  • 70% ethanol

Measurement parameters and instrument settings

The “NanoQuant Nucleic Acid Quantitation” control bar can be easily selected using the SparkControl software. Two options are available for blanking: average blanking and individual blanking (default setting).

For average blanking, the average value of all positions employed for the blanking procedure is used for correcting the sample measurement values. For individual blanking, the sample values measured in each sample position are used for subtracting the blank values for the same position.

The workflow panel of the SparkControl software, appearing after blanking followed by the sample measurement, is shown in Figure 2.

Figure 2. Application stripe after blanking.

The SparkControl software automatically measures all wavelengths for the quantification of nucleic acids, where 310 nm is used as a reference wavelength for internal correction. The measurement results contain a full spectrum from 200 to 1,000 nm and also the 260/280 and 260/230 ratios.

DNA quantification and purity checks

The plate was cleaned using blank solution, according to the Quick Guide for the NanoQuant Plate, and then measurements were made [1]. Measurements were taken for 16 replicates of four different concentrations, as shown in Table 1.

Table 1. DNA concentrations used for quantification and purity checks

Dilution

Concentration (μg/ml)

A

50

B

25

C

12.5

D

6.25

 

OD260 linearity

A Phage Lambda-DNA dilution series was used to measure the measurement linearity at 260 nm. As summarized in Table 2, the Phage Lambda-DNA was serially diluted 1:3 in EDTA buffer. For each concentration, eight replicates were averaged and blanked and this is followed by plotting the corrected average OD260 values.

Table 2. Dilution series of Phage Lambda-DNA

Dilution

Concentration (μg/ml)

A

300

B

100

C

33.3

D

11.1

E

3.7

F

1.4

 

Results

DNA quantification and purity checks

Table 3 lists the average values after referencing and blanking, clearly showing that the DNA concentrations measured using the SparkControl software correlate well with the theoretical sample concentration.

In the case of pure DNA samples, the 260/230 and 260/280 measurements were well within the expectation. There is good measurement uniformity (% CV across all 16 replicates), ranging from 1.44 to 8.54%. In this application, the theoretical detection limit for DNA measured was measured to be below 1 μg/ml.

Table 3. Results of the DNA quantification and purity check

DNA conc. (μg/ml)

%CV

260/280 ratio

260/230 ratio

Theoretical

Measured

50

50.4

4.73

1.87

2.03

25

26

1.44

1.88

2.07

12.5

12.1

5.53

1.89

2.09

6.25

6.9

8.54

1.90

2.09

 

A DNA spectrum between 215 and 300 nm for one DNA sample after blank reduction is shown in Figure 3. The resulting spectrum, in addition to being of high quality, provides further information about sample purity. This information can be used to help optimize lab productivity and increase the efficiency of analysis.

Figure 3. DNA spectrum (50 μg/ml sample) after blank reduction measured in the NanoQuant Plate using 2 μl sample volume

OD260 linearity

The measurement linearity at 260 nm employing the DNA concentrations summarized in Table 2 is shown in Figure 4. The R2 of the dilution curve is normally above 0.999 between OD values of 0 and 3.5, ensuring a wide measurement range, preventing the need for dilution of high concentration samples, and thus, further increasing the assay’s effectiveness and productivity.

Figure 4. Measurement linearity at 260 nm

Conclusion

Fitted with High Speed Monochromators, the Spark® multimode microplate reader offers extremely fast scanning capabilities by enabling a high linearity for DNA quantification as well as full spectrum acquisition in less than five seconds per sample. A preconfigured ‘one-click’ software application for purity determination and quantification of nucleic acids provides support to this ingenious hardware design.

The study results show that the combination of the new Spark® reader and the NanoQuant Plate offers an efficient, reliable tool for the quantification of nucleic acids in low volume samples. The exclusive software application combines purity checks (260/280 and 260/230 ratios) and nucleic acid quantification with a rapid scan of the full spectrum from 200 to 1,000 nm, and delivers more useful data for additional analysis.

References

  1. Quick Guide NanoQuant Plate No.30035094 Rev No. 1.4

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: Jul 14, 2018 at 7:13 PM

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