UV Absorbance Measurements of DNA in Microplates

UV absorbance measurements in microplates became possible only five years ago when Molecular Devices (Sunnyvale, CA, USA) introduced the SPECTRAmax 250, the first UV-Visible microplate spectrophotometer. Microplates have since become very popular for making UV measurements of DNA, RNA and protein concentrations because of their higher throughput compared to traditional cuvettes. Despite their huge popularity, many people experience difficulty when adapting assays to them. Especially in the UV range, microplate assays require an awareness of the optical properties of the microplate materials and more attention to technique than do traditional cuvettes to get accurate absorbance results. Absorbance measurements made through microplates are subject to path length variability and are vulnerable to interference from surface effects at the air/liquid interface. Samples have different absorbance values when read in microplates and cuvettes, unless the microplate values are normalized for optical path length. Finally, modern microplate spectrophotometers are designed with increasingly narrow beams to accommodate smaller microplate wells and thus are much more vulnerable to spurious readings caused by particles than older, wide-beam instruments. Lack of attention to these details is the most frequent cause of difficulty in adapting assays to microplates. The following report provides guidelines for optimizing DNA measurements in microplates. It also gives an example of expected results if the guidelines are followed and illustrates the effect of ionic strength on DNA absorptivity. UV Transparent Microplates. Quartz microplates are commercially available (Hellma Cells, Forest Hills, NY, USA), but are expensive (>$1000). Standard polystyrene microplates do not transmit light below approximately 300 nm. However, there are at least three UV transparent plastic microplates available (Figure 1). The UVPlate (Corning-Costar, Cambridge, MA, USA) and the UV-Star (Greiner, available from E & K Scientific Products, Saratoga, CA, USA) have good transmission down to approximately 215 nm. The UVMax (Polyfiltronics, available from Phenix Research Products, Hayward, CA, USA) is usable down to 240 nm. The UV-Plate and UV-Star are comparable to a quartz microplate for DNA/RNA analyses. UVMax microplates have a higher background, but good results can be obtained by prereading the plates and subtracting the background on a wellby-well basis. Recommendations for optimizing DNA absorbance measurements (3,4). First, use clean microplates and particlefree solutions. Particles can cause artifactual spikes in absorbance values by as many as 0.3 absorbance units in modern narrow-beam instruments. Remove particles by filtering buffers (e.g., pore size ≤5 μ). If needed, remove dust by blowing out the microplate with clean dry air. Cover microplate if it is not to be read immediately. Second, note absorbance values of buffer blanks (and include blanks at least in duplicate). The UV-Plate and UV-Star microplates vary slightly between lots, but the mean A260 value for a water-filled plate should be between 0.046 and 0.060 with a SD ≤0.002. The mean A260 value for a water-filled quartz plate should be 0.03–0.04 with a SD ≤0.002. Buffer-containing blanks may have higher values. If the blank values do not fall into the expected range, the most likely cause is a dirty microplate, particles in the wells or a defective microplate. Third, if your procedure calls for a small sample volume (1–20 μL) and a diluent, pipet the small volume into the well first, followed by the larger volume. Mix well with the pipettor or by vibrating the microplate. Fourth, recognize that absorbance depends on the meniscus curvature as well as the sample volume. The optical path length (and therefore the absorbance) of a microplate sample depends not only on its volume but also on the degree of meniscus curvature. The meniscus can be nearly flat (water or buffer in untreated microplates) or highly curved (detergent-containing sample or surface-treated microplate). The resulting difference Benchmarks