Nucleic acid quantification is a vital step in many biochemical workflows. Accurate RNA and DNA quantification improves experiment repeatability and can save significant amounts of time. Knowing the precise concentration of nucleic acid samples can also help with resource management, thus saving costs and high-value materials of limited availability. But carrying out RNA or DNA quantification precisely can be complicated.
Several methods exist for RNA and DNA quantification, and not all of them were created equally. At DeNovix, we specialize in nucleic acid analysis via fluorescence and through UV-Vis spectrophotometry. Real-time PCR is another option available to life scientists looking to quantify DNA samples. But which is the best DNA quantification method?
Real-Time Polymerase Chain Reaction
Real-time PCR, sometimes referred to as qPCR, is one of the most common RNA and DNA quantification methods in use today, owing to its sensitivity, specificity, and dynamic range. It involves repeated phases of thermal cycling that cause nucleic acid chains to fragment and polymerize. Fluorescent reporters stain the fragments and a detection instrument measures the relative fluorescent units (RFU) of the sample as a function of the cycle profile. Though sophisticated, real-time PCR quantification of RNA and DNA has its drawbacks.
The cost of ownership is often prohibitive, while multiplexing can be extremely challenging. Additionally, real-time PCR demands a high level of operator expertise and a significant amount of time for assay preparation and running.
A UV-Vis spectrophotometry operates on the Beer-Lambert law, which correlates the absorption of light to the characteristics of the material through which that light is traveling. Concentration is one of the specific characteristics that can be interrogated using light of specific wavelengths and known pathlengths – the distance light travels through the sample. UV-Vis spectrophotometry is a powerhouse in RNA and DNA quantification, but performance varies between instruments. Those with fully automated pathlength adjustment and lamps with a continuous spectrum across a wide range across the ultra violet and visible spectrum (190 – 840 nm) typically deliver the best results and greatest flexibility.
Nucleic acid samples are assayed with fluorescent reagents. A fluorometer illuminates the sample with specific excitation wavelengths from one or more light sources and the emitted light is captured and converted to Relative Fluorescent Units (RFU’s). It is possible to correlate the concentration of unknown samples through comparison against standard curve of know concentrations. This method relies on the intrinsic binding specificity of fluorophores enabling the development of kits highly specific for a single analyte in the presence of contamination.
Absorbance and Fluorescence can be considered contrasting but complimentary methods for sample quantification. Absorbance using microvolume UV-Vis is very quick, accurate and able to indicate any potential contamination within a sample. It is also possible to measure over a wide dynamic range without the need for dilutions e.g. the DS-11 Series will measure in a range of 0.75 ng/µL and 37500 ng/µL dsDNA. Fluorescence assays are highly specific for a single molecule and have enhanced sensitivity – DeNovix dsDNA assay kits enable quantification to 0.5 pg/µL dsDNA.
At DeNovix, we recognize the benefit of combining both absorbance and fluorescence in a single instrument which champions precision, sensitivity, specificity and ease-of-use.