The Take3™ Multi-Volume Plate has proven to be a flexible tool, providing the ability to perform absorbance measurements over a wide range of applications and sample volumes in a BioTek microplate reader. Micro-volume determinations of up to sixteen 2 μL samples of extracted, undiluted DNA, RNA and protein-based on native absorbance measurements have been demonstrated with precision and accuracy suitable for downstream applications such as sequencing, qPCR and protein analysis1-3.
The Take3™ Multi-Volume Plate alsolends itself to colorimetric assays, where the color-producingreagent is added to the plate subsequent to sample addition. Themicrospots of the Take3 plate act as both reaction and measurementvessel, which simplifi es workfl ow and conserves sample. It hasbeen demonstrated that quantification accuracy is signifi cantlyimproved using this workflow relative to workfl ows requiringreagent incubation in a separate vessel4.
Here we extend the utility of theTake3 plate by demonstrating micro-volume fl uorescent assays ofDNA using the Synergy™ Multi-Mode Microplate Reader product lineand Quant-iT PicoGreen® reagent to extend the linear dynamic rangeof ds-DNA quantifi cation to low ng/mL concentrations.
Material and Methods
Materials
A Quant-iT™ PicoGreen® dsDNA reagent Kit was obtained from LifeTechnologies, Molecular Probes Division (Eugene, OR, PN-P7589). ThePicoGreen reagent was freshly prepared by diluting 15 μL reagentwith 2 mL of TE buffer (~ 133X) and stored in the dark for nolonger than 2 hours. DNA standards were prepared from concentratedstock herring sperm dsDNA (Sigma, PN-A3294) in TE buffer(tris-EDTA, pH 7.0).
Protocols
All Picogreen reactions and measurements were performed in theTake3 plate. A protocol was defined similar to that alreadydemonstrated with the in-situ BCA protein assay5. The protocol andTake3 plate map are demonstrated in Figure 1. Briefly, 2 μLaliquots of standard or unknown sample were added to the respectivemicrospots of the Take3 plate. The diluted solution of PicoGreenreagent was then loaded to each microspot in a 1:1 ratio (2μL/microspot) using a manual 8-channel pipettor with mixing. Theblank consisted of 2 μL of TE buffer reacted with 2 μL of Picogreenworking reagent in the appropriate microspots of the Take3plate.
After closing the top lid of theTake3 plate, the reaction was incubated for 5 minutes atroom-temperature (~ 22º C), in the dark, then read on either aSynergy H4, using both monochromators and spectral filters or on aSynergy HT Microplate Reader. All measurements were made with Gen5™Software.
Figure 1. Take3 plate map depictingpositions and concentrations of Lambda dsDNA standards andpositions of up to four unknown samples. This map provides a6-point calibration curve run in duplicate over rows A-F, whichcovers the in-situ micro-volume assay working range of 10- 1,000ng/mL.
Subsequent to each analysis, bothtop and bottom slides were wiped 2-3 times with a methanolsaturated laboratory wipe to remove any residual reagent andair-dried.
Instrument Settings and DataAnalysis
Both dual-monochromator and filterbased optical systems of the Synergy™ H4 Hybrid Multi-ModeMicroplate Reader and top reading fl uorescence of the Synergy HTwas used to quantify the DNA solutions. Monochromator bandwidths of9 nm were used in all measurements: the excitation monochromatorwas set to 495 nm and the emission monochromator to 526 nm. It wasalso determined that a z-axis vertical offset of 6 mm providedhighest signal-to-noise ratio when using the monochromators.Spectral filters used by both Synergy H4 and HT were 485 nm, 20 nmbandwidth for excitation and 528 nm, 20 nm bandwidth for emission.All Synergy measurements relied on the Xenon fl ash lamp as theexcitation light source (high lamp energy was selected for spectralfi lter determinations on measurements). The automatic sensitivityadjustment feature in Gen5 was used for all data collection withscaling using high DNA concentration wells F2:F3 (1000 ng/mL) to60,000 relative fluorescence units with 255 measurements per datapoint.
Take3 plates with whiteTeflon-printed slides defi ning the microspots can be used withSynergy monochromator optical systems as the cross-section of thedetection volume defi ned by the optical system is smaller in sizethan the microspot dimension. For spectral filter optical systems,signifi cant fl uorescent background was evident which reduced theanalytical performance at lower DNA concentrations to unacceptablelevels. This issue was alleviated by using black Tefl on-printedslides which served to signifi cantly reduce background. Allmeasurements made in this work utilized black Tefl onprintedslides.
All DNA measurements were blankcorrected. Signal to noise (S/N) determinations at each DNAconcentration were made using blank subtracted mean fl uorescencedivided by the standard deviation of the DNA fl uorescence signal.Standard curves were fi t using a linear function in GraphPadPrism.
Results & Discussion
The 6-point standard curve used forthe PicoGreen assay provided a working range of 10 – 1000 ng/mLDNA. Figure 2 demonstrates the linearity of the standard curvesobtained with each optical system. Regardless of the optical systemused all data collected resulted in a least means squared linearregression analysis with coeffi cient of determination (R2) valuesof > 0.999 for n=3 experiments. It is evident from the slopes ofthe standard curves that the spectral fi lter systems provideslightly higher sensitivity than the monochromators.
Figure 2. Least means squared linearregression analysis of standard curve generated from data acquiredon both the monochromator and spectral fi lter optical systems ofSynergy H4 and Synergy HT. Error bars (too small to view) representSEM (n=3 experiments).
Figure 3 demonstrates S/Ndeterminations at each of the DNA concentrations used in thestandard curve for all the optical systems used. It is evident thatsignifi cantly higher S/N is available with the monochromatoroptical system at low DNA concentrations (≤ 100 ng/mL). This is dueto the lower background signals associated with the smallercross-section of the detection volume provided by themonochromators. The increased light throughput associated withspectral fi lter systems tends to provide higher S/N at higher DNAconcentrations (≥ 100 ng/mL).
Figure 3. Signal to Noise (S/N)determinations at each DNA concentration used in the standard curvefor each of the Microplate Readers and respective optical systems.S/N is defi ned in the Materials and Methods section. Error barsreflect cumulative error (standard deviation) in blank and DNAmeasurements.
The availability of 16 microspots onthe Take3 provides the ability to generate standard curve andunknown sample data at the same time. By performing the assay andmeasurements in the same vessel quantifi cation errors caused bydifferences in incubation time, temperature and sample manipulationare minimized. Using the Take3 plate map defi ned in Figure 1, theaccuracy of two samples of known concentration, one at low DNAconcentration (25 ng/mL) and another at higher concentration (500ng/mL) were quantifi ed using the monochromator optical system ofthe Synergy H4. Excellent accuracy was observed at bothconcentrations: + 1.6% accuracy at 25 ng/mL DNA (over-estimation)and – 3.0% accuracy at 500 ng/mL DNA (underestimation). Similarperformance was seen across the Synergy Microplate Reader productline.
Conclusion
The Take3 plate is a useful tool forperforming fluorescent micro-volume assays such as lowconcentration DNA quantifi cation down to low ng/mL concentrations.This effectively extends the dynamic range for DNA quantificationby approximately three orders of magnitude relative to measurementsinvolving native absorbance at A260. The in-situ protocol wheresamples, standards and PicoGreen regent are successively added tothe Take3 plate provides the means to simplify workflow, conservesample and reagent and improve the accuracy of determination. TheTake3 plate can be used across the Synergy Microplate Readerproduct line for micro-volume analysis.
References
1. Brescia, P. and Banks, P. Multi-Volume Analysis of Nucleic Acidsusing the Epoch™ Spectrophotometer System, Winooski, (VT), BioTekInstruments, Inc., Nov,2009.
2. Brescia, P. and Banks, P.Analytical Performance of Nucleic Acids Micro-Volume Quantification Using the Epoch™ Spectrophotometer System, Winooski,(VT),BioTek Instruments, Inc., Dec, 2009.
3. Brescia, P. and Banks, P.Analytical Performance of Epoch™ Multi-Volume SpectrophotometerSystem for Protein Quantifi cation, Winooski, (VT), BioTekInstruments, Inc., Jan, 2010.
4. Brescia, P. and Banks, P. In-situMicro-Volume Bicinchoninic Acid Protein Assay, Winooski, (VT),BioTek Instruments, INC., Feb, 2010.