Case 1: How do I proceed with low-concentration sample solutions?
UV-Vis spectrophotometry is not very sensitive – in order to obtain accurate and precise data, sample concentrations should therefore not be allowed to fall below a certain concentration threshold. For dsDNA, this lower limit is approximately 1 µg/mL. Even if the lower limit of detection of the photometer has not been reached, the impact of measurement inaccuracy in this range is considerable, and resulting values may be prone to substantial variation. Fluorometric analysis, being 1000-fold more sensitive than the absorbance method, is capable of quantifying sample solutions of extremely low concentrations very accurately.
Case 2: How can I perform accurate quantification despite sample contamination?
In addition to quantification of nucleic acids at 260 nm, UV-Vis spectrophotometry offers the option of determining sample purity by employing additional wavelengths (230 nm, 280 nm, 320 nm), or a wavelength scan, respectively. Data analysis will allow conclusions regarding the presence of substances such as salt, proteins or solid particles. If, on the other hand, the absorbance spectra are too similar, as is the case for the different nucleic acids, absorbance alone will not be able to distinguish between, for example, RNA und DNA. In this case, fluorescence measurements will allow the specific quantification of analytes, without interference from other molecules.
On which principle is this method based?
Fluorescence measurements take advantage of the phenomenon that certain molecules are capable of emitting energy in the form of light. For the detection of biomolecules such as nucleic acids and proteins, a fluorescent dye is selected that will bind to the analyte with the highest possible specificity. The binding of the reagent PicoGreen® to dsDNA is a classic example. Following excitation of the complex using light of a specific wavelength (approximately 480 nm for PicoGreen), the fluorophore will emit light of lower energy, i.e. of a longer wavelength (approximately 520 nm for PicoGreen) (figure 1). The light is measured as relative fluorescence (RFU), where the intensity of the fluorescence is proportional to the concentration of the sample.