Nucleic acid quantification, encompassing double stranded DNA (dsDNA), single stranded DNA (ssDNA) and RNA quantification, is a staple process in life science applications requiring knowledge of sample concentration prior to downstream experiments. Both types of nucleic acid are typically quantified using one of two optical technologies: ultraviolet-visible (UV-Vis) spectroscopy, or fluorescence measurements.
Accurately quantifying nucleic acid concentrations is critical for downstream applications such as next-generation sequencing, transfection, PCR and cloning. Such applications frequently require a target range of nucleic acid concentration and purity for optimum performance and inaccurate determination of concentrations can cause higher variability or assay failure.
The Principals of Fluorometric Analysis
Fluorometric analysis provides sensitivity at around 1,000 times higher than absorbance-based methods. It is also a feature of fluorescence assays that they are highly specific to the analyte of interest compared to those absorbance.
Quantification of DNA, RNA or Protein through fluorometric methods require the binding of analyte-specific fluorophores to the sample of interest. The sample/fluorophore complex is illuminated with a specified excitation wavelength from a light source. The fluorophore emits light over a characteristic spectrum and the amount of emitted light is proportional to the amount of analyte in the sample. The light is captured and recorded as relative fluorescent units (RFUs). The concentration of unknown samples can be understood through the correlation to a standard curve of known concentration samples.
Fluorometric Analysis Equipment from DeNovix
The DS-11 Series and QFX fluorometer from DeNovix are at the forefront of fluorometric analysis equipment, being more sensitive and reproducible than any other instrument in its class. The QFX is a powerful solution for fluorometric applications where improved sensitivity or specificity is required while the DS-11 Series combines microvolume and cuvette absorbance with fluorescence for total sample quantification method flexibility.
DeNovix Fluorometers have four fluorescent channels for quantification enabling compatibility with all major quantification assays as well as future-proofing labs for future assay developments.
Using the DeNovix Fluorescence quantification assays, researchers can quantify dsDNA between 0.5 pg/µL and 4000 ng/µL and a single RNA assay quantifies in the range of 250 pg/µL to 1500 ng/µL. Each assay is well-suited solution for quantification of degraded or low concentration samples, even with common contaminants present.