PHARMACEUTICAL application note Nucleic Acid Quantitation in Microplates HansWilly Mueller, Ph.D. J. Fenton Williams, Ph.D. Introduction Many molecular biology applications require the determination of ss-, dsdna, or RNA concentration in various samples. Most commonly, UV spectroscopy or Fluorescence spectroscopy is the preferred method of choice. Most simple and most convenient is UV spectroscopy, because nucleic acids have a characteristic UV absorbance spectrum, which can be used or quantitation without further sample derivatization and the technique is non-destructive; the sample can be used again after measurement. Nucleic Acids show a typical absorbance maximum at 260 nm in the UV range of the spectrum (see Figure 1). This wavelength is therefore used for quantitation of a DNA sample. According to Beer-Lambert s law, the absorbance reading is proportional to the concentration. Typical proportionality constants are 50 for dsdna, 33 for ssdna and 40 for RNA. Absorbance readings of the nucleic acids obtained in a 1 cm cell multiplied by the respective proportionality constant will give the concentration in µg/ml. Most common Fluorescence methods use PicoGreen dye for dsdna determination, OliGreen for ssdna determination, and RiboGreen for RNA determination. Both dyes are measured in a fluorescence spectrometer with 485 nm excitation wavelength and 535 nm emission wavelength. A big advantage of Figure 1. UV Absorbance Spectrum of a 50 µg/ml dsdna sample, showing typical absorbance maximum around 260 nm and minimum at 230 nm. UV Absorbance at 260 nm fluorescence methods is their selectivity. This means that the above methods can be used for selective detection of any single nucleic acid type in the presence of one or more of the others. Fluorescence methods are also more sensitive than UV absorbance measurements. The sample, however, has to be treated with a fluorescent reagent for measurement and thus no longer behaves as a native molecule in subsequent procedures. Since fluorescence is so sensitive, however, it is usually possible to analyze only a tiny fractional portion of the entire sample. For sensitivity comparison of Fluorescence methods and UV Absorbance methods see Table 1. Fluorescence ssdna 100 ng/ml-100 µg/ml OliGreen 1 ng/ml-1 µg/ml dsdna 150 ng/ml-150 µg/ml PicoGreen 250 pg/ml-1 µg/ml RNA 120 ng/ml-120 µg/ml RiboGreen 1 ng/ml-1 µg/ml Table 1. Comparison of measurement ranges for DNA quantitation using UV Absorption spectroscopy and Fluorescence spectroscopy. UV measurement range is based on measurement at 260 nm in a 1 cm cuvette. Look to us. And see more.
Sample throughput in conventional UV and Fluorescence spectrometers is very limited and relatively high sample volumes, 0.3 to 3 ml, are needed for standard cuvette measurements. For measurements in 96 well microplates only 50-300 µl sample is needed and higher sample throughput can be achieved. Newer applications therefore use the universally standardized 96 well format for DNA preparations (for example DNA Miniprep-Kits). Also oligonucleotide synthesis is getting more and more productive, requiring fast and simple measurement of synthesis yields. These technology advancements thus require a different approach for DNA quantitation than standard UV measurements with conventional spectrometers. UV and Fluorescence microplate readers are ideal for these higher throughput situations, because 96 low volume samples can be measured in much less than a minute. All measurements described here were obtained with the Perkin-Elmer HTS 7000 BioAssay Reader. This novel system allows simple software selection of both through-well absorbance measurements and top or bottom fluorescence measurements in any style microplate. The ability to do both types of measurements (UV absorbance and fluorescence) in one instrument has many advantages. The most obvious advantage is the flexibility to optimize different kinds of tests in one instrument. In addition it is also very useful for fluorescence measurement to know the absorbance of the fluorescent dye. The absorbance at excitation wavelength should be below 0.05A for linear fluorescence measurement. 384 (16x24) well format, with 80 µl maximum volume, is rapidly gaining acceptance and can be accommodated by the HTS 7000 BioAssay Reader. Fluorescence measurements are usually performed in white or black opaque microplates. Excitation light is directed via an optical fiber toward the sample in the well. The emitted fluorescent light from the reporter dye is captured by another fiber and directed to the detector. For special applications such as averaging multiple readings in cell function assays, epifluorescence measurement is also done from the bottom of special plates with clear well bottoms and opaque sides. UV measurements must be done with transparent microplates. Because DNA is measured at 260 nm, special plates are needed, made from an UV transmitting material. Conventional polystyrene microplates, which are commonly used for colorimetric ELISA tests, cannot be used for DNA measurements because they do not transmit UV light. For the UV measurements, special commercially available UV transparent plates were used. These plates have excellent UV transmission properties. As can be seen from Figure 2, these plates remarkably transmit 85% of 260 nm light. Converted to absorbance, this means that an empty well will give an A260 reading of only 0.07A, which is excellent for low noise DNA measurement and can be easily subtracted as a blank reading. Different than in a conventional UV/Vis spectrophotometer using a cuvette, the pathlength of the sample in a microplate will naturally vary, depending on the sample volume in the well, the slope of the sides of the well and the shape of the bottom of the well. The optical pathlength (b) in microwells usually need not be known exactly, but is precisely controlled by the typically excellent liquid handling reproducibility (see Figure 3). Since pipetting accuracy and precision is very good, the volume accuracy and precision achieved Using Microplates A wide range of multi-well microplates are available on the market. The most commonly used type is the 96 (8x12) well plate with 350 µl maximum volume, but other formats are used as well. A trend towards plates with more wells is evolving due to high sample throughput needs of drug screening assays. So the Figure 2. Transmittance spectrum of a single well of a Corning Costar UV microplate, showing usable wavelength range between 210 nm and 800 nm.transmittance of an empty well, measured at 260 nm is about 85% (= 0.07A). Typical Standard Deviations across the empty plate are < 5 ma for empty wells. Figure 3. Optical pathlength in microwell is dependent on the sample volume. Theoretical pathlength may be calculated as b = Vol, with r = radius of the well (mm) π*r 2 Vol= sample volume (µl). 2
by modern pipetters will directly translate to pathlength accuracy and precision. Precision of mulitichannel pipettes is typically < 0.8% for 50 µl or < 0.2% for 300 µl volume. The accuracy is about ±1.5% for 50 µl and ± 0.6% for 300 µl volume. This variable pathlength in the wells of microplates has the advantage that assay detection sensitivity can be controlled. For more concentrated samples, less volume may be used, whereas for diluted samples, the well may be filled completely. The variable pathlength thus offers a convenient means of sensitivity adjustment without requiring special cuvette-sets with various pathlengths. For practical experiments, however, it is not recommended and not necessary to calculate the pathlength geometrically from the volume. For all volumes, the pathlengths are typically less than predicted by geometry due to the meniscus shape. The meniscus shape may be different, depending on plate type, buffer or possible detergent used. Also, because well shape (wall slope and bottom shape) may vary from plate to plate a geometric calculation of pathlength is not advisable. The difference between calculated (theoretical) and optically determined pathlength is shown in Table 2. The theoretical pathlength was calculated from the dimensions (6.4 mm diameter) of a well of a 96 well plate and various volumes using the equation b = Vol π*r 2. Volume Calculated Pathlength Optically measured (µl) (mm) pathlength (mm) 50 1.6 1.4 100 3.1 2.9 150 4.7 4.4 200 6.2 5.8 250 7.8 7.4 300 9.3 8.6 Table 2. Calculated pathlength compared with actually measured pathlength. Pathlength as a function of sample volume filled into the wells of a Costar UV Plate. Sample:Various volumes of 50 µg/ml DNA solution, measured at 260 nm. The experimental determination was done with a 50 µg/ml DNA solution of which the respective volumes were pipetted into the wells of a 96 well Costar UV Microplate (Catalogue Number 3635). Ten replicates were measured per volume in the HTS 7000 Bio Assay Reader at 260 nm. The same sample was measured in a standard 1 cm cell in a conventional UV spectrometer. In both cases blank correction was done with the same volume of buffer. From the Absorbance values measured in the plate reader the optical pathlength was calculated, based on the A (1 cm) readings from the conventional UV spectrometer. Calibration For practical DNA quantitation in microplates, it is not necessary to know, compute or correct for the pathlength. According to Beer- Lambert s law, A=K*c, and there is a linear relation between absorbance reading and concentration. The proportionality factor K is the product of the molar extinction coefficient and the pathlength, neither of which have to be known independently. When working with a reference solution, a simple single standard calibration is sufficient and the result will be correct, independent of the pathlength (or volume) used. A pathlength correction is not required and therefore this one standard method is very convenient and reliable. It is good laboratory practice to do a complete calibration curve once, to check the linear measurement range of the spectrometer system and to assure that the compound to be measured obeys Beer-Lambert s law. This is shown in Figure 4 and Figure 5. A 200 µl aliquot of a serial dilution of 250 µg/ml ds DNA was pipetted in five replicates into the microwells of a 96 well plate. Figure 4 shows the Figure 4. HTSoft Plate map for dsdna calibration with 11 standards, 5 replicates each. 3
Absorbance Absorbance dsdna Absorbance Calibration Curve y=0.0110618* x +0.0320148 d=0.0071726 r=0 999617 3 2.5 2 1.5 1 0.5 dsdna Absorbance Calibration Curve y=0.0113174* x +0.0131474 d=0.00268626 r=0 999936 3 2.5 0 0 50 100 150 200 250 300 ds DNA concentration (µg/ml) 2 1.5 1 0.5 0 0 50 100 150 200 250 300 ds DNA concentration (µg/ml) Figure 5. Calibration curve obtained from calibration with 11 standards of dsdna (200 µl per well). Linear calibration curve fit. Calibration curves were obtained with the Plate map from Figure 4. Figure 5a: linear curve fit with all standards. Figure 5b: Linear curve fit with standards above 2.5 A excluded. Volume in Absorbance in Approximate dsdna Microplate Microplate with Absorbance in Concentration HTS 7000 1 cm cell 300 µl 2.5 A 2.8 A 140 µg/ml 250 µl 2.5 A 3.4 A 170 µg/ml 200 µl 2.5 A 4 A 200 µg/ml 150 µl 2.5 A 5.5 A 275 µg/ml 100 µl 2.5 A 8 A 400 µg/ml 50 µl 2.5 A 16 A 800 µg/ml Table 3. Maximum concentration of dsdna, which can be measured in a microplate with the HTS 7000 BioAssay Reader. Maximum absolute amount of dsdna is about 40 µg. For comparison the theoretical absorbance values are listed as they would be measured (if possible) in a 1 cm cell in a conventional UV Spectrometer. Usually, the range of a 1 cm cuvette-based UV spectrometers is 0 to 3A. plate map, and Figure 5 shows the resulting calibration curves. The linear regression calibration curve shows an excellent correlation coefficient of r=0.999617, although very high absorbing samples are included in the analysis. The HTSoft instrument control and evaluation software calculated this statistical value from the curve fit of the measured standards. The software will also allow optimization of the calibration curve, by offering selective exclusion of known incorrect data points and recalculating another curve without these points. The two calibration curves from Figure 5 illustrate this data editing capability. When the two highest concentrations with absorbances above 2.5A are not used, the correlation coefficient improves incrementally to r=0.999936. Both curves are equally useful for any typical DNA quantitation in molecular biology labs. In a 200 µl sample, it is possible to measure dsdna up to or above 2.5 absorbance units with the HTS 7000, depending on the precision required. 2.5A linear measurement means that dsdna concentrations of 800 µg/ml can be measured with HTS 7000 BioAssay Reader, when 50 µl sample is filled in the wells. As it can be seen from Table 3 these high absorbance values correspond to very high concentrated DNA solutions which will be impossible to measure with a 1 cm cell in a conventional UV spectrometer without dilution. An undiluted 800 µg/ml dsdna solution would result in a theoretical absorbance of about 16A when a 1 cm cell is used. The maximum absolute amount of dsdna, which can be measured with the HTS 7000 BioAssay Reader without dilution is about 40 µg. Quantitative DNA Measurement in Practice When it is once proven that a measurement system gives linear response to the DNA concentration, it is not anymore required to perform extensive calibrations. A simple universal concentration factor is not 4
Figure 6. HTSoft Plate map for single point calibration. Blank and 200 µg/ml standard are measured with 4 replicates. recommended, because DNA samples from different origins have unique molar extinction coefficients, depending on their base composition. A quick calibration is therefore recommended. Single point calibrations are sufficient within the proven linear range of the system. As long as the sample volume per well is kept constant for a set of samples, one single standard can be applied. This single standard is quickly prepared and should contain the same type of DNA, diluted in the same buffer system as the sample. Actual measurement of this single standard should be done in 2-4 replicates (see Figure 6). HTSoft will calculate the average and the standard deviation of the replicates. The single point calibration will give accurate results, because the standard is measured under identical conditions as the samples. Especially it is a very elegant way to compensate for pathlength effects. Also, other parameters such as volume, temperature, buffer system and preparation steps etc., are the same for the standard and the samples and their effects are automatically compensated for in a single calibration point system. This way of measuring DNA concentration is much better than measurement with known pathlength. When pathlength is known, a precise extinction coefficient must be known in order to obtain correct results. The correct extinction coefficient, however, may vary with solvent, buffer salts or ph. So it really is easier and strictly most relevant to prepare and to measure one standard. Knowledge of pathlength and extinction coefficient is not required for correct results. Even when samples in groups with different volumes in one plate have to be measured, this is easily possible using one standard for each group. HTSoft allows up to 12 different experimental groups to be defined on a single 96 well plate. Once an assay has been established and reproducibility is acceptable, HTSoft can support the common requirement of needing to apply the standard curve from the plate with the single point standard to other plates with just samples. An application with increasing importance is DNA mini preparation. DNA preparation kits facilitate simple and rapid DNA preparation (Qiagen Qiaprep and Qiawell ). These kits use the 96 well format and allow preparation of plasmid DNA within less than an hour. The eluate can be collected in an UV transparent microplate and then it is ready for direct quantitation. Eluates from these preparation kits contain up to 20 micrograms of DNA in a 75 µl Figure 7. HTSoft Concentration results from DNA miniprep. 5
eluate, which can be measured in 30 seconds using the HTS 7000 BioAssay Reader. The pathlength at this volume is large enough to allow reproducible absorbance measurements, as long as air bubbles on the meniscus surface are avoided. Also the measurement range is ideal: With 20 µg DNA in the 75 µl eluate, the absorbance at 260 nm will be slightly above 1, which is well within the linear measurement range of the HTS 7000. The following brief protocol describes how to determine dsdna concentration in 75 µl eluates obtained directly from Qiagen DNA minipreps. 1. Preparation of standard solution. You can either use a defined dilution of the DNA type to be prepared with the Miniprep. Recommended concentration is 200 µg/ml: Dissolve 1 mg DNA in 5 ml 10 mm Tris-Cl buffer (ph 8.5) (e.g. Sigma D-3644; 1 mg preweighed DNA that is readily soluble may be used). This dsdna standard solution has a concentration of 200 µg/ml. 2. Setup of Plate reader method on the HTS 7000. This method will use absorbance endpoint mode with 260 nm as measurement wavelength and an HTSoft plate map as shown in Figure 6. This is an example where wells A1-D1 are filled with 75 µl buffer for blank measurement and wells A2-D2 are filled with 75 µl standard solution (= 4 replicates each). The rest of the plate is filled with 75 µl sample (eluate), obtained from the Qiagen miniprep columns. In this example four replicates are used for blank and the single standard. Absorbance 0.75 Single Point Calibration for dsdna Quantitation 0.5 0.25 0 0 50 100 150 200 Figure 8. HTSoft single point calibration graph for concentration determination. 75µL sample was used. 3. Plate Filling. Wells A1-D1 will be filled with 75 µl buffer solution, Wells A2-D2 will be filled with 75 µl DNA standard solution. All other wells of the microplate will be filled with 75 µl sample eluate from the miniprep columns. 4. Plate measurement. Measurement of the entire plate will take about 25 seconds. After this, the results will be displayed. Concentration results (see Figure 7) are based on single point calibration in wells A2-D2. The respective calibration graph is shown in Figure 8. Conclusion For labs with many samples to process, the HTS 7000 Bio Assay Reader in the UV absorbance mode at 260 nm offers a very quick and convenient way to measure DNA concentrations on a daily basis. The [ds DNA ] µg/ml popular 96 well plate format now offers affordable UV-transparency capabilities and therefore obviates the need for more complex and cumbersome quartz cuvette sipper and autosampler systems. The universality of the microplate format assures a smooth transition for DNA samples being processed in parallel at molecular biology labs. High sample throughput, low sample volume and wide concentration detection range are the main advantages. The HTS 7000 is of special interest for DNA quantitation in DNA Minipreparations. HTSoft provides final DNA concentration results from any array of samples and blanks. As long as pipetting is precise and reproducible, knowledge of exact pathlength in the well is unimportant because a single standard calibration will produce reliable and accurate results for all samples. Visit our Website at www.perkin-elmer.com. U.S.A.,The Perkin-Elmer Corporation, 761 Main Avenue, Norwalk, CT 06859-0010 Tel: (800) 762-4000 or (203) 762-1000. Fax: (203) 762-6000 Perkin-Elmer is a registered trademark of The Perkin-Elmer Corporation. D-5647 August 98 Printed in U.S.A. 1998 The Perkin-Elmer Corporation Look to us. And see more.