Nucleic Acid Quantitation in Microplates

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 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. P H A R M AC E U T I C A L