Techniques such as PCR and next-generation sequencing (NGS) have changed how researchers work with nucleic acids, particularly with regard to scale and throughput. It is key to be able to accurately quantitate the amounts of nucleic acid starting material to truly leverage these methodologies. Microplate readers provide an option for researchers to rapidly and accurately quantify nucleic acid levels.
Nucleic acid quantification is traditionally performed by spectrophotometric absorbance measurement at 260 nm in a 1-cm quartz cuvette. That type of measurement offers a high degree of precision and accuracy, but does not meet the need for high-throughput, miniaturized assays. Microplate readers are now used to quantify DNA and RNA in multiplex assay with volumes as low as a few microliters.
Microplates are used to process dozens or hundreds of samples within an experimental run. They are applicable to any imaginable type of assay, including immunoassays, enzymatic assays, cell-based assays, and more, as well as for quantification of nucleic acids and proteins. For nucleic acid quantification purposes, tool providers offer UV-transparent microplates.
In addition to UV absorbance, fluorescence intensity and luminescence also can be used for quantifying nucleic acids. Fluorescence offers a more sensitive result than absorbance, especially when concentrations are low. Excitation and emission wavelengths depend on the dye used in the sample. A calibration curve consisting of a dilution series of known concentrations of a known molecule is generated and used to determine the concentrations of unknown samples being analyzed.
Multimode instruments
Although a single-mode instrument may serve perfectly well for a laboratory with simple, straightforward needs, many researchers are turning to multimode instruments that offer a choice of modes for detection.
The advantages of a multimode instruments include the ability to take many different types of measurements in a single, plate-based format economically and with a small bench footprint.
Many vendors offer modular multimode plate readers, so that only the capabilities that are desired need to be purchased. Researchers can choose to upgrade the instruments based on their experimental needs.
For example, the Thermo Scientific Varioskan LUX microplate multimode reader includes several measurement technologies, according to Tuula Jernstrom, marketing manager for sample preparation and analysis at Thermo Scientific. In addition to absorbance and fluorescence intensity, as well as fluorescence resonance energy transfer (FRET), the instrument can measure luminescence, AlphaScreen/AlphaLISA and time-resolved fluorescence.
Accuracy
Accurate DNA quantification is essential for many applications in molecular biology, such as cloning, NGS and qPCR. Those methods depend on equal, normalized amounts of DNA input. Errors at this stage will intensify errors downstream, when DNA is often amplified via PCR reactions. Accuracy and low detection limits are also significant to the emerging use of cell-free DNA (cfDNA) as a biomarker in liquid biopsies, in areas like noninvasive sample diagnostics, organ-transplant monitoring, and other clinical applications.
Low concentrations of DNA, such as from precious sample materials like cfDNA, can present additional challenges for researchers.
Poor-quality DNA or RNA also can be difficult to quantify accurately.
Some products available that address these accuracy challenges include Thermo Scientific’s high-sensitivity dyes (Quant-iT kits) and the KingFisher sample-purification system for extracting high-quality nucleic acids from challenging starting materials.
Path length
In transitioning from quartz cuvettes to microplates, the path length of the measurement is significantly changed. Because an accurate path length is crucial in nucleic acid concentration determination, switching from a fixed horizontal path format to a vertical path through an extremely small droplet can introduce error.
Path-length calibration can be carried out for microplates using a set of standard solutions of known concentration. The correct path length can then be calculated using Beer’s law.
An alternative to the process of path-length calibration is a product like Tecan’s NanoQuant plate, which is designed to have a path length of exactly 0.5 mm, when 2 μl of sample are pipetted onto the sample spot.
“You only have to pipette 2 μl of the samples on each of the spots. You close the lid. Then it forms [a] 0.5-mm column. This is the so-called path length. This is exactly what the NanoQuant plate has. Each of the 16 spots has exactly 0.5-mm path length,” says Michael Fejtl, market manager for Tecan’s detection products.
BioTek Instruments’ Take3 Micro-Volume Plate also accommodates a 2-μl sample and uses a nominal vertical path length of 0.5 mm. According to Lenore Buehrer, a senior product manager with BioTek, when Take3 is used in one of BioTek’s compatible microplate readers, Gen5 software makes the path-length correction automatically. “The nucleic acid concentrations are automatically converted and reported in 1-cm equivalent values,” Buehrer shares.
Wavelength precision
A technical consideration similar to the path-length challenge is wavelength accuracy. Nucleic acid purity is calculated using the ratio of absorbance at 260 nm to 280 nm. The primary contaminant in nucleic acid samples is protein, which has a natural absorbance peak at 280 nm. If the tolerance of the wavelength setting on the instrument is slightly off, it could result in significant error in the reading at 280 nm. That’s because 280 nm is on the slope of the overall spectrum.
“In our readers, we strive to provide accurate measurements through high tolerance in wavelength selection. We have an accuracy specification of 2 nm,” says Paul Held, an application scientist with BioTek.
Protein contamination of DNA or RNA samples can sabotage experiments at subsequent stages, Held says, explaining that “proteins can interfere with experiments down the road. If the presence of protein is not accounted for, it can result in a false value for nucleic acid quantification.”
And Tecan’s Spark instrument has wavelength accuracy of 0.3 nm, according to Fejtl.
Software
Although there are many microplate readers on the market that will provide adequate data, usability can vary. The software interface can become an important differentiator among products. The Thermo Scientific SkanIt microplate-reader software is designed to be intuitive across a range of applications and skill levels.
According to Jernstrom, SkanIt software includes a path-length correction function that simplifies scanning. “For evaluating purities, both instruments allow spectral scanning of nucleic acids, and there are ready-made sessions in the internal software of Multiskan GO for easy measurements using cuvettes. Further, the intuitiveness and simplicity of SkanIt software allows setting up these photometric assays within seconds, thus speeding up researchers’ workflows,” says Jernstrom.
On the software side, Tecan offers one-click applications with its instruments. “You basically have to pipette the sample into the Nanoquant plate, place the Nanoquant plate in the reader, and the whole measurement is executed automatically,” says Fejtl. “This is where software is going. Single-app, single-click, ease-of-use feature.”
Beyond the basics
Beyond the typical considerations of accuracy and precision, features such as speed or the ability to customize the workflow for a particular experiment can make a big difference to researchers in terms of usability of the instrument.
For example, according to Fejtl, the speed of an instrument is not only a matter of convenience but can affect accuracy. Fejtl calls Tecan’s Spark monochromator the fastest instrument of its kind on the market. “When you’re using low-volume samples, the issue is evaporation. That’s particularly the case with other systems, of course. Readouts of a monochromator can take up to 10 seconds. Only 2 μl are used in such plates. A fast monochromator helps get better data quality by avoiding evaporation,” he says.
Other advanced features for microplate readers include automated liquid handling and environmental controls for cell-based assays.
Microplate readers have become a ubiquitous piece of laboratory equipment, a 21st century equivalent to the single-cuvette spectrophotometer that once occupied a bench in every life-science laboratory. Many options and upgrades are now available to improve or enhance the accuracy and usability of these instruments. Path-length calibration, wavelength accuracy, speed, and well-designed software should be taken into consideration when purchasing a microplate reader. In addition, microplate readers can offer multiple modes of detection and features like environmental controls and automation to streamline workflow.
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