Low Volume DNA and RNA Quantification using NanoQuant Plate™

The detection and quantification of minute amounts of nucleic acids are needed in many recent forensic and genetic tests. The most common technique to calculate nucleic acid concentrations involves measuring the absorbance at 260 nm (A260). The absorbance values from 260 nm and 280 nm measurements (260 / 280 ratio) are compared to test the purity of the RNA or DNA.

Dye-labeled probes are needed for array hybridization experiments and real-time PCR assays. A vital parameter for obtaining comparable results is the degree of labeling of the RNA or DNA probe with a specific dye (labeling efficiency).

Using Tecan’s new NanoQuant Plate, the purity and concentration of nucleic acids can be measured for up to 16 samples at the same time. In addition, this new tool enables determining the efficiency of the nucleic acid labeling with dyes.

Tecan’s NanoQuant Plate

Since a sample volume of only 2 μl is needed for these applications, the NanoQuant Plate is a suitable tool for the latest applications in molecular biology.

Material and methods

Instruments

  • Infinite M200 Quad4 Monochromator detection system (Tecan Austria)
  • Infinite F200 filter-based detection system (Tecan Austria)

NanoQuant Plate

Figure 1 shows the NanoQuant Plate featuring 16 (2 x 8) separate sample positions with specially designed quartz optics (Tecan Austria).

Figure 1. Tecan’s NanoQuant Plate. The figure shows the Nano-Quant Plate with its lid closed.

For accurate positioning within the reader, every NanoQuant Plates is supplied with optimized plate definition files (pdfx).

Reagents

  • 20x TE (Invitrogen, CA)
  • Nuclease free water (Fluka BioChemika, Switzerland)
  • lambda-DNA, 100 μg/ml (Invitrogen, CA)

Nuclease-free water was used to dilute phage lambda-DNA in 1x TE to the final concentrations of 5, 10, 20 and 50 μg/ml.

Measurement parameters and settings

Table 1 shows the measurement parameters for Infinite F200 and Infinite M200 instruments used for DNA quantification and purity check. The absorbance was set at 310 and 340 nm as a reference wavelength for internal correction for the Infinite M200 and the Infinite F200 instruments, respectively.

Table 1. Measurement parameters for Infinite M200 and Infinite F200 with Tecan i-control software

Measurement parameters for Infinite 200 instruments

Parameter

Setting

Absorbance

Bandwidth

Absorbance

260 nm

5 nm

280 nm

Bandwidth

5 nm

Settle time

0 ms

Number of reads

25

 

The introduction of an application tab allows the control bar “Quantify Nucleic Acid” to be selected, among other items, for easy handling in Tecan’s i-control software.

Figure 2 shows the strip as it appears in the workflow pane of the i-control software after blanking and the subsequent sample measurement.

Figure 2. Quantify Nucleic Acid Strip as it appears in the i-control Software after blanking.

Before initiating the measurements, the NanoQuant Plate cleaning was performed as outlined in the Quick Guide for the NanoQuant Plate, prior to the measurements [1]. After blanking on the NanoQuant Plate quartz positions, the samples were pipetted. A multi-channel pipette was employed for sample application.

Results

For all 16 measurement positions, the measurement results of referencing and blanking, nucleic acid quantification, purity check, and labeling efficiency are automatically displayed in Excel®. Figure 3 shows the result of one well (A1) for demonstration.

Figure 3. Excel sheet output of the measuring results for position A1. The raw data for 260 nm, 280 nm and Cy5 absorbance are shown in the left columns. Calculations to get the values for nucleic acid con-centration, purity check and dye concentration are shown in the right columns.

DNA – Quantification and purity check

The NanoQuant Plate was used to measure DNA samples ranging from 5 to 50 μg/ml. Table 2 lists the average values after referencing and blanking.

Table 2. Absorbance measurements of DNA samples from 5 μg/ml to 50 μg/ml. Results are blank corrected using the NanoQuant Plate

DNA [μg/ml]

5

10

20

50

Abs 260 [OD]

0.0047

0.0082

0.0184

0.0489

Abs 280 [OD]

0.0023

0.0042

0.0098

0.0259

Ratio 260/280

2.07

1.95

1.88

1.88

 

The NanoQuant Plate was used to determine the detection limit for nucleic acid quantification, which was found to be 1 μg/ml or 1 ng/μl.

Labeling efficiency

The NanoQuant Plate was used to measure an amplified cDNA probe after labeling the probe with Cy5. An absorbance scan from 230–700 nm was generated using the Infinite M200 scanning option to collect more data about Cy5 labeled probe (Figure 4).

Figure 4. Absorbance scan of Cy5 labeled cDNA probe. Scan from 230 – 700 nm, bandwidth 5 nm, raw values are plotted (non-referenced, non-blanked) for generating the curve. Note that Cy5 has two absorbance peaks (605 and 649 nm).

Conclusion

The increasingly smaller volume of sample used in many modern molecular biological applications makes it increasingly difficult to determine the concentration of nucleic acids in modern molecular biology. For instance, forensic analysis sometimes uses only a few cells to obtain DNA probes for genetic fingerprinting.

Tecan’s NanoQuant Plate can provide reproducible and sensitive quantification of nucleic acids as well as their purity check. The labeling efficiency is a vital parameter to validate results during quality control of oligonucleotide labeling reactions in array hybridization experiments employing dye-labeled probes and in real-time PCR assays. The labeling efficiency of nucleic acid probes can be easily determined using the NanoQuant Plate.

The NanoQuant Plate uses a unique mathematic algorithm to reference each measurement position used for calculations, thus delivering accurate and reliable results.

Literature

  1. Quick Guide NanoQuant Plate, 2008

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: May 31, 2017 at 4:18 AM

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