Nucleic acid quantification is a key step in molecular biology workflows, particularly for advanced techniques such as Next-Generation Sequencing (NGS). It is also critical for more routine tasks like setting up ligation reactions for basic cloning experiments. Virtually every downstream assay that requires input DNA or RNA requires that you know how much you have.
Quantification methods are limited by their sensitivity, accuracy and ability to distinguish between the various types of nucleic acids such as dsDNA, ssDNA and RNA.
There are three commonly used methods for quantifying nucleic acids:
- UV absorbance
- Fluorescent dye-based quantification
- qPCR (also called real-time or quantitative PCR)
QUANTITATING NUCLEIC ACIDS BY ABSORBANCE MEASUREMENTS (UV ABSORBANCE)
Perhaps the most common quantitation method is UV-spectrophotometry (also called absorbance spectroscopy). This technique takes advantage of the Beer-Lambert Law: an observation that many compounds absorb UV and visible light at unique wavelengths. For UV-visible spectrophotometry, light is split into its component wavelengths and directed through a solution. Molecules in the solution absorb specific wavelengths of light. For a fixed path length, the absorbance of a solution is directly proportional to the concentration of the absorbing molecule. DNA, for example, has a peak absorbance at 260nm (A260).
Quantitating nucleic acid by absorbance is user-friendly, quick and easy, providing a rapid assessment of concentration and purity, but it has some limitations. UV-absorbance is not a sensitive measurement; the lowest detectable concentration of DNA using absorbance measurement is 2ng/µl. So, while absorbance quantitation will suffice for applications like cloning, it will not be sensitive enough for downstream applications like NGS, qPCR or ddPCR (digital droplet PCR) that may use DNA at extremely low concentration as input.
Additionally, DNA is not the only molecule that absorbs light at 260nm. Organic compounds including proteins, phenols, guanidine and other nucleic acids absorb light at this wavelength. You can use this fact to get an idea of the purity of your sample by measuring absorbance at 260nm and 280nm (where protein absorb light), for instance. Such a measurement will estimate the relative amount of nucleic acid compared to protein. However, it will not give you information about the integrity of your nucleic acids, and it cannot distinguish among dsDNA, RNA or ssDNA molecules or their size. Because these non-template compounds can contribute to the absorbance reading, they can cause over-estimation of your sample concentration.
FLUORESCENT DYE-BASED QUANTIFICATION
Fluorescent dye-based quantitation uses specially designed DNA binding compounds that bind in a specific manner to nucleic acids. When excited by a specific wavelength of light, only dye in the bound state will fluoresce (unbound dye does not fluoresce), and that fluorescent signal is directly proportional to the amount of nucleic acid in the sample. These aspects of the technique contribute to low background signal and therefore the ability to accurately and specifically detect very low quantities of DNA in solution, even the nanogram quantities used in NGS applications.
Fluorescent dye-based quantitation is only slightly more complex than a simple absorbance measurement. A standard curve is generated with known concentrations of DNA, and the concentration of DNA in the experimental sample is interpolated from the curve. If using a single-tube fluorometer, such as the Quantus™ Fluorometer (below), only a blank and one standard are used to calibrate the instrument. For fluorescent quantitation, this measurement is possible over a dynamic range for those concentrations that are common for NGS applications (0.01-400ng/µl).
Fluorescent dyes are now available in a truly “add-and-read” format. Dye solution and as little as 1µl of sample are mixed and measured on a fluorometer.
Fluorescent dyes provide several advantages for nucleic acid quantitation. Low background signal due to lack of fluorescence without binding provides greater sensitivity. Additionally, fluorescent dyes can be optimized to bind to specific species of nucleic acids. For instance, the QuantiFluor® dyes have been optimized to bind primarily to either dsDNA, ssDNA or RNA, giving you specificity in your nucleic acid measurements that is not possible with absorbance quantitation.
Quantitation of Nucleic Acids Using qPCR
In real-time, quantitative PCR (qPCR) the amplified product is measured after each PCR amplification cycle. The amplification curve illustrates the accumulation of the product as PCR progresses and can be divided into three basic parts: the baseline, or the initial reporter fluorescence, measured before product formation is detected; the exponential phase; and the plateau phase, the stage in which the rate of product formation diminishes.
qPCR curves are generally viewed on a linear scale to more clearly see the different parts of the curve. Doing so allows you to easily define and identify several points. The amplification threshold is the level of fluorescence that distinguishes between background and a true amplification product. The Cq is the cycle number at which the sample signal passes the amplification threshold, indicating a positive signal. The ∆Rn is the total change in signal after amplification is complete.
Using qPCR, a standard curve is generated for a known sample by plotting the Cq versus the concentration for a known sample. Concentrations of unknown samples are extrapolated from the standard curve based on Cq value. Amplification efficiency is also derived from the standard curve and is a function of the slope. Relative quantitation compares the Cq of the experimental sample to the Cq of the control sample and will be adjusted based on the efficiency of the reactions.
RT-qPCR (reverse transcription followed by qPCR) is a powerful technique for gene expression analysis. It can be used quantitate or determine the absence of a given transcript or other RNA molecule. qPCR and RT-qPCR are powerful, specific techniques for Quantitating nucleic acids. They are highly specific and provide information about intact nucleic acids.
However, qPCR requires a knowledge of experimental design, setup and analysis that is not needed in simpler quantitation techniques. qPCR assays often require 30-60 minutes to execute, in contrast with the relatively rapid methods such as absorbance spectroscopy. Furthermore, samples such as blood, food, soil or faeces may contain inhibiting compounds that can prevent accurate measurement by qPCR. These limitations can be overcome in part by using a nucleic acid purification system designed to remove inhibitors and qPCR master mixes that are resistant to inhibitors.