Applications - Application Notes
Analytical Performance of Nucleic Acid Micro-Volume Quantification Using the Epoch™ Spectrophotometer System
Authors: Peter Brescia and Peter Banks, BioTek Instruments, Inc., Winooski, VT
Nucleic acids such as DNA and RNA are derived from a variety of biological sources for potential use in downstream applications such as sequencing and gene expression analysis. Here we demonstrate the analytical performance of the Epoch™ Spectrophotometer System and especially the Take3™ Multi-Volume Plate with regard to linear dynamic range, limit of detection, precision and accuracy using micro-volume samples to verify its broad applicability to nucleic acid quantification. Comparative data was derived on Epoch Spectrophotometer System and NanoDrop: performance was shown to be equivalent.
Accurate determination of molecular concentrations is prerequisite to the use of purified nucleic acids for a multitude of downstream applications. Quantification is routinely accomplished by spectrophotometric analysis at 260 nm for nucleic acids in a UV transparent vessel. Measurements were historically made with quartz cuvettes that have a fixed pathlength of 1 cm and are typically associated with high precision and accurate measurements. Recently, BioTek has developed the Epoch™ Spectrophotometer System that is capable of accurately measuring up to 16 samples with volumes as low as 2 µL (Figure 1) using the Take3 Multi-Volume Plate. The Take3 plate provides a nominal 0.5 mm pathlength allowing measurement over a broad range of concentrations. 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 nucleic isolation methods – yield is dependent on sample type, size and what sort of nucleic acid is being isolated. The Take3 plate is also capable of accommodating two BioCells and a standard cuvette extending the range of quantifiable sample concentrations to sub ng/µL by the extension of pathlength to 1 cm.
Figure 1. Take3 Multi-Volume Plate shown with BioCell™ in place (a). Two additional locations are available for a second BioCell and a standard cuvette. Sample volumes as low at 2 µL can be loaded on 16 microspot locations using either a single- or multi-channel pipettor.
Here we show the analytical performance of the Epoch Spectrophotometer System for micro-volume analysis in terms of detection limit, linear dynamic range, precision and accuracy for both dsDNA and RNA.
Material and Methods
Linear Dynamic Range
All double-stranded DNA (dsDNA) and RNA standards were created by preparing a 1:2 serial dilution series of a concentrated stock of herring sperm dsDNA or yeast (Saccharomyces cerevisiae) RNA, respectively, in TE buffer (tris-EDTA, pH=7.0). Epoch Spectrophotometer System micro-volume data was obtained with undiluted standard samples using the Take3 plate. The Take3 plate was calibrated prior to use to determine pathlength correction values for each microspot. Each standard concentration was loaded 5-times at each microspot location on the Take3 plate using an 8-channel manual pipettor. Optical densities were read at 260, 280 and 320 nm resulting in 80 replicate measurements on each instrument. BioCell™ data was acquired using either undiluted or, for higher concentration samples, a 20-fold dilution of standard in TE or MilliQ water. NanoDrop micro-volume data was determined from replicate measurements of the same nucleic acid standards. All sample measurements were background corrected using a TE buffer blank or MilliQ water, where appropriate. All concentrations depicted are based on a 1 cm pathlength and 50 ng/µL/OD for DNA; 40 ng/µL/OD for RNA.
Limit of detection 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 each microspot of the Take3 plate was determined from 10 measurements of reloaded blank solution. As with both DNA and RNA measurements made in the Linear Dynamic Range determinations outlined above, 260 nm signals were corrected bichromatically at 320 nm.
Results & Discussion
Linear Dynamic Range & Accuracy
Herring sperm dsDNA standards were prepared as a 12 point 1:2 serial dilution series resulting in concentrations ranging from ~ 4 to 3,000 ng/µL. Micro-volume measurements using both Take3 and NanoDrop were compared to those taken using the BioCell placed in the Take3 plate and read on the Epoch microplate reader and appear in Figure 3. It is apparent that the linear dynamic range of the Epoch Spectrophotometer System for micro-volume analysis is three orders of magnitude, which adequately covers the yield and concentration ranges of most DNA isolation kits (Table 1).
Figure 3. dsDNA standard curve using dilutions of a purified herring sperm sample. Abscissa data is considered actual [dsDNA] as measurements were conducted with 1 cm pathlength using BioCell™. Ordinate data is micro-volume determinations using Epoch™ Spectrophotometer System and NanoDrop. The straight line is through the origin and has a slope of 1.000, which is considered a perfect equivalence between micro-volume and BioCell data. Inset is an exploded view of low dsDNA concentrations.
Table 1. Expected [DNA] in isolate from some commonly used commercially available DNA isolation kits. Yields are provided as expected results from supplier product literature.
Linear regression analysis was also performed on the data presented in Figure 3. Both micro-volume determinations demonstrated near perfect straight line correlations (R2 ≥ 0.9998) and slopes of 1.020 and 0.967 for Epoch Spectrophotometer System and NanoDrop, respectively. The slope can be used as a measure of accuracy relative to 1 cm pathlength measurements across the [DNA] range presented in Figure 3. Thus Epoch Spectrophotometer System shows an average percent difference of 2.0% relative to 1 cm pathlength determinations across three orders of magnitude [DNA]; NanoDrop, 3.3%.
Yeast RNA standards were prepared as 10 point 1:2 serial dilution series resulting in a concentration range of ~ 4 to 2,400 ng/µL. Micro-volume measurements using both the Epoch Spectrophotometer System and NanoDrop were compared to those taken using the BioCell placed in the Take3 Multi-Volume Plate and read on the Epoch microplate reader (Figure 4). It is apparent that the linear dynamic range of the Epoch Spectrophotometer System is three orders of magnitude, which covers the yield and concentration of most RNA isolation kits (Table 2).
Figure 4. RNA standard curve using dilutions of a purified yeast RNA sample. Abscissa data is considered actual [RNA] as measurements were conducted with 1 cm pathlength using BioCell. Ordinate data is micro-volume determinations using Epoch Spectrophotometer System and NanoDrop. The straight line is through the origin and has a slope of 1.000, which is considered a perfect equivalence between micro-volume and BioCell data. Inset is an exploded view of low RNA concentrations.
Table 2. Expected [RNA] in isolate from some commonly used commercially available DNA isolation kits. RNA yields from tissue samples start with ~ 25 mg of material; 106 HeLa cells were used to generate 20 µg of RNA. Yields are provided as expected results from supplier product literature.
Linear regression analysis was also performed on the RNA standard curve data presented in Figure 4. Both micro-volume determinations demonstrated perfect straight line correlations to 4 decimal places (R2 = 1.0000) and slopes of 0.9900 and 0.9975 for Epoch Spectrophotometer System and NanoDrop, respectively. Thus, Epoch Spectrophotometer System shows an average accuracy of 1.0% relative to 1 cm pathlength determinations across three orders of magnitude [RNA]; NanoDrop, 0.25%.
It is evident from Tables 1 and 2, that the concentration of isolated nucleic acid will vary over more than two orders of magnitude depending on the sample type, amount, elution volume chosen, nucleic acid to be isolated and isolation kit used. The precision of the quantification may differ across such a large dynamic range of concentrations. Figures 5 and 6 demonstrate the precision of replicate measurements of dsDNA from Figure 3 and RNA from Figure 4 respectively, spanning the concentration range depicted in Tables 1 and 2. It is evident that even at low nucleic acid concentration, precision is ≤ 2.5% CV and averages ≤ 1.5% CV, suitable for accurate quantification.
Figure 5. Precision measurements expressed as a %CV from each of the individual 16 microspots at dsDNA concentrations as determined by BioCell. A: 35.7 ng/µL; B: 266 ng/µL; C: 1050 ng/µL;
Figure 6. Precision measurements expressed as a %CV from each of the individual 16 microspots at RNA concentrations as determined by BioCell. D: 38.2 ng/µL; E: 304 ng/µL; F: 1220 ng/µL.
Limit of Detection
Table 3 shows the magnitude of the standard deviation for each microspot on the Take 3 plate. All noise signals are below 0.001 OD. The absorbance signal representative of the detection limit can be computed from the average standard deviation from the 16 microspots multiplied by a factor of three. This detection limit is 0.0011 OD.
Table 3. Standard deviation in the blank signal for each of the 16 microspots of the Take3 plate. These data multiplied by a factor of three are representative of an absorbance equivalent to the detection limit for each of the 16 microspots.
The limit of detection for both dsDNA and RNA can be computed from the data generated for the linear dynamic range determinations in conjunction with this detection limit expressed as an absorbance signal. The lowest concentration of herring sperm DNA used in the standard curve was determined to be 4.7 ng/µL by using the 1 cm pathlength BioCell; for RNA, it was 4.3 ng/µL. The average background corrected absorbance signal generated by the 16 microspots was determined to be .0042 OD for dsDNA; for RNA, it was .0053 OD. Therefore, the detection limits for dsDNA and RNA are 1.2 and 0.90 ng/µL, respectively.
The analytical performance of the Epoch Spectrophotometer System is characterized by a detection limit of 1 ng/µL for the micro-volume spectrophotometric determination of nucleic acids at 260 nm. The linear dynamic range for both dsDNA and RNA extends more than three orders of magnitude beyond this to over 2,500 ng/µL. Accuracy relative to 1 cm pathlength determinations with BioCell display a percent difference of ≤ 2.0% over the full concentration ranges of dsDNA and RNA. Precision over this concentration was typically below 1% CV, but no more than 2.5% CV at low nucleic acid concentrations. This wide concentration range is typical of the yields from current nucleic isolation methods, so good linearity and precision over this range demonstrates broad applicability to nucleic acid quantification. Equivalent performance was seen between micro-volume determinations with Epoch Spectrophotometer System and NanoDrop.