How to quantify and qualify Nucleic Acids samples?
Workflows in the molecular biology are often time consuming. By creating control steps within the workflow high quality results can be maintained. Different ways of Quantification and Qualification of input materials are available depending on the needs acquired for your specific molecular platform. By measuring both the concentration as the purity of the nucleic acid samples, technicians can decide to discard samples based on certain quality criteria.
There are two main approaches used by scientists to quantitate nucleic acids in a solution. These are spectrophotometric, absorption spectrum based, and quantification, absorption spectrum based, and fluorometric quantification, based on fluorescent tagging of nucleic acids.
UV-Vis absorbance is the primary method for determining the concentration and purity of DNA or RNA. A UV-Vis spectrophotometer measures the intensity of light transmitted through a sample compared to a reference measurement of the incident light source.
Both single- and double-stranded DNA strongly absorb ultraviolet light with a peak absorbance wavelength of 260nm. The signal is measured by spectrophotometers or spectrometers. A UV-Vis spectrophotometer is equipped with one or more channels of an incident light source that covers the full ultraviolet-visible (UV-Vis) electromagnetic spectrum (190 – 840nm).
Fluorometers are used for precise quantitation of nucleic acids and proteins in samples. This requires the to be detected material to be bound to a specific fluorescent agent. Tagged samples are subsequently loaded into the instrument in a polypropylene tube, which determines the level of fluorescence that can used for calculating the concentration of the labeled-molecules. This way they indirectly measure the concentration of double- and single-stranded DNA (dsDNA and ssDNA), ribonucleic acid (RNA), and proteins in a solution.
The use of DNA-binding dyes allows more specific measurement of DNA than spectrophotometric methods allows, providing biomolecular detection at sensitivities of up to 0.0005 nanograms per microliter (ng/μl). The downside of this method is that no information about the purity of the sample can be provided.
Wat is the difference between quantification and qualification?
Nucleic Acid quantification is generally performed to determine the average concentration of DNA or RNA in a sample prior to proceeding with downstream applications. It can have a negative effect when concentrations are too low or too high. When the DNA or RNA concentration in a sample will be too low, changes are that the downstream application simply won’t work. Luckily molecular techniques are getting more and more sensitive nowadays, so the amount of nucleic acids necessary are decreasing. This means that the techniques used for quantification of Nucleic Acids in the samples needs to be more sensitive as well. Ideally, you should combine the strengths of an absorption spectrum, in sight in purity next to an indication of the concentration, and fluorometric detection, enabling highly sensitive absolute concentration measurements.
In some cases it also matters when the concentration is too high and output can’t be detected anymore. Also, when working with precious patient material it is important to use as less material as possible.
Besides Quantification, Qualification plays a role as well. When the quality of DNA or RNA in the sample solution is not optimal, the results from the downstream application can be interfered. The quality is equal to the purity of the sample, and this can affect the quantification of a sample.
The influence of the sample purity on quantification
Contaminations in purified DNA can lead to problems ranging from inaccurate quantification or incorrect determination of the starting material to problems in downstream assays due to contaminant interference. In these cases it is recommended to use a spectrophotometer for qualification of your samples, since it is not possible to detect the purity using the fluorescence method and a fluorometer for quantification.
Measuring the absorption of a sample at 260 nm divided by the absorbance measured at 280 nm is a widely used method for quantifying DNA concentration. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present.
To detect other possible contaminants, the absorption is also measured at 230 nm and 320 nm. Absorbance around 230 nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260 nm to 230 nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. As a guideline, the A260/A230 is best if greater than 1.5.
A reading at 320nm will indicate if there is turbidity in the solution, another indication of possible contamination.
All of these absorbance measurements together help determine the quantification and qualification of nucleic acids in samples.