Microvolume Nucleic Acid Quantification
July 2022
Quantification of isolated biomolecules is routinely accomplished by spectrophotometric analysis in a UV transparent vessel. Quantification at 260 nm for nucleic acids is made possible by the absorption of ultra-violet light by chromophores contained in the nucleic acids. Measurements were historically measured with quartz cuvettes that possess a fixed path length of 1 cm and are typically associated with high precision and accuracy of measurement. Consistent with these methods is the use of the Beer-Lambert Law that relates physical property of light absorption by molecules to concentration within the sample by the following equation:
where ε refers to the molar absorptivity or extinction coefficient of the analyte, l is the path length (cm) and c is the concentration of the analyte. For double stranded DNA, the commonly accepted average extinction coefficients at 260 nm is 0.020 (ng/µL)-1cm-1 and for single stranded RNA = 0.025 (ng/µL)-1cm-1. The assessment of the purity of a nucleic acid sample is often performed by a procedure commonly referred to as the A260/A280 ratio which refers to two spectrophotometric measurements made at these defined wavelengths. Pure nucleic acid samples would have an A260/A280 ratio of 1.8-2.0.
Agilent BioTek developed novel accessories capable of accurately measuring 16 to 48 samples, with volumes as low as 2 µL, to aide in the preservation of precious sample (Figure 1).
The Take3 and Take3 Trio microvolume plates provide a nominal 0.5 mm fixed path length allowing measurement over a broad range of concentrations. Each plate is compatible with the Cytation, Synergy, and Epoch readers for microvolume absorbance and fluorescence quantification. Concentrations can be measured from dilute, low ng/µL samples as well as samples in the 1000’s of ng/µL range. This wide concentration range is typical of the yields from current biomolecular isolation methods and the measurements can be made in a microplate spectrophotometer. The recently launched Take3 Application allows convenient, preprogrammed, and customizable methods to assess both quantification and purity of nucleic acids.
Featured Applications
Microvolume Purity Assessment of Nucleic Acids Using A260/A280 Ratio and Spectral Scanning
A common method to determine the purity of biomolecules from sample isolates is by use of a spectrophotometric ratio using absorbance measurements at wavelengths of 260 nm and 280 nm. The A260/A280 ratio provides a rapid indication of protein contamination in nucleic acid isolates and less commonly, nucleic acid contamination in protein isolates. In addition, UV-Vis scanning can identify contamination from chemical additives used in the isolation procedure. This application note assesses this capability in microvolume analysis using the Agilent BioTek Take3 microvolume plate.
Analytical Performance of a Higher Throughput Microvolume Microplate Accessory for Microplate Spectrophotometer Platforms
Accurate determination of molecular concentrations is a prerequisite to the use of purified biomolecules for a multitude of downstream applications. Quantification is routinely accomplished by spectrophotometric analysis. A novel accessory is described here which incorporates the ability to directly measure up to forty-eight microvolume (2µL) samples for direct quantification and two vertical 1 cm path length cells, in a standard microplate sized format.
Product Spotlight
Take3 Microvolume Application
The new Take3 microvolume app is a stand-alone app with a modern interface that allows quick and easy nucleic acid and protein quantification without the need to work within Agilent BioTek Gen5 or Gen6 software.
The intuitive interface has five easy steps from samples to results:
- Load blanks and samples
- Select a protocol
- Assign and blank microwells
- Assign and read samples
- View and export your results
Simple plate management allows the user to add multiple Take3 and Take3 Trio plates. Pathlengths can be imported or added manually for each plate in the library.
Users have the option to use preloaded protocols as well as the ability to add custom protocols for user defined nucleic acids and proteins.
The application preferences have a number of blanking, reference wavelength selection and pathlength correction options.
Tek Tips
Limit of Detection
The limit of detection (LOD) is typically defined as the analyte concentration that can provide a signal that is three-fold higher than the noise (standard deviation) of the background signal. The standard deviation in the blank signal for the 48 microspots of the Take3 Trio plate and Agilent BioTek microplate reader was determined and LODs calculated for DNA and RNA.
The LODs were determined to be 6 mOD and 4 mOD for dsDNA and RNA, respectively. Using standard mass coefficients based on a 1 cm path length of 50 ng/mL/ OD for DNA and 40 ng/mL/OD for RNA the LODs are 2 ng/µL for dsDNA and 3.2 ng/µL for RNA. Given very strict cleanliness and pipetting skill requirements needed to obtain accurate measurements approaching these limits it is recommended to work above the calculated limit.
When working with very small pathlengths and analyte volumes a small speck of dust or smudge on the vessel surface can have a very significant effect on the absorbance measurement. Therefore, it is recommended that working concentrations be 5x-10x the LOD. When working below 5x- to 10x the LOD, measurements should be verified using a 1 cm pathlength vessel if accuracy is required to be less than 10% error. Most biological preparations for downstream applications are well above the LOD required for accurate quantification by microvolume measurements.
Using the Take3 Application for blank and sample readings on the Take3 Trio and viewing results
When using a Take3 Trio plate it is possible to blank on each sample well or a range of wells and use the blank average. This is defined in the Application Preferences. It is recommended that when quantifying samples at or near the LOD to blank on each sample well for greater accuracy. The default setting uses an average with a 10% CV validation limit that can be adjusted as needed. A very clean Take3 plate can easily provide less than 3% CVs across all wells during blanking with either filtered, double-distilled water or analyte buffer.
Samples and blanks can be assigned when using a range of wells to blank.
Samples are assigned using the plate layout tab, located at the bottom of the screen, after blanks are read if blanking on all wells. There are now four plates available for viewing at the top of the screen when the plate layout tab is selected. The first plate will be all positions with blanks.
Plates 2-4 will show the plate map of each individual slide with 16 spots. Additional plates can be added once the read is complete and added to the plate count at the top of the screen up to 10 plates.
When viewing results using the Summary tab only plates 2-4 will have sample results.
Plate 1 will have no sample data and will present the following message "No samples on this plate." Additional plates can be added once the read is complete and added to the plate count at the top of the screen. When measurements are completed, all data, including well blanking data, can be easily exported to a tab delimited text file using the Export All Summary Data button in the upper right corner of the window.
Microvolume Nucleic Acid Quantification Resources
Application Notes
- Analytical Performance of Nucleic Acid Micro-Volume Quantification Using the Epoch Spectrophotometer System
- Microvolume Determination of Labeling Efficiency Using Spectral Scanning Analysis in a Standard Microplate Reader
Video
Agilent BioTek Take3 and Take3 Trio Microvolume Plates and Take3 App
Webinar
LAMP Assays: Kinetic characterization using a Multimode Microplate Reader
Speaker: Peter J. Brescia, Jr., MSc, MBA
Wednesday, August 03, 2022 | 12 PM EDT
Isothermal nucleic acid amplification tests, such as Loop-mediated isothermal amplification (LAMP) have emerged over the past 20 years as an alternative to polymerase chain reaction (PCR). The use of LAMP assay technology spans a range of applications including its use as an important diagnostic tool in clinical applications, and food quality control and environmental monitoring. LAMP has gained traction for its ability to detect nucleic acid sequences without specialized equipment using a variety of detection modes. LAMP assays are typically run as an end point reaction in PCR tubes. In this larger format, observation for positive/negative determinations can be made manually via visualization of a colorimetric change due to pH. This methodology lends itself to adaptation for automated kinetic monitoring in a higher-density microplate format at low volumes.
In this webinar we will present:
- Background and benefits of LAMP compared to conventional PCR
- Guidelines and tips for adapting LAMP assays to a kinetic microplate-based format
- Microplate reader settings for LAMP assay optimization
