Nucleic acid quantitation is a critical step in sample preparation that helps ensure optimal performance of downstream assays. A common misconception is that all quantitation methods are alike and have similar accuracy. But in fact, concentration estimations can differ depending on what method is used. Thus, it is crucial to understand the basis of each method so you can choose the most suitable one for your need. This article describes the principles, advantages and limitations of the three most common methods for DNA or RNA quantitation: spectrophotometry, fluorescence and real-time PCR.
Fluorescence dye-based quantitation methods take advantage of this mechanism by employing dye molecules that have been designed to preferentially bind a given species of nucleic acid. When a dsDNA dye, for example, is excited by a given wavelength of light, only dye in the dsDNA-bound state will fluoresce. As the dye binds to the target nucleic acid, fluorescent quantum yield increases as a function of shift in fluorophore molecular geometry. This aspect of the fluorescence quantitation technique, in conjunction with preferential dye:target binding, results in a low background signal, high accuracy and specificity, making it ideal for quantitation of low-level nucleic acid samples (Figure 4).
For this type of assay to be quantitative, a dilution series of sample of known concentration is used to create a standard curve. A fluorometer is used to read and record the relative fluorescence units (RFUs) for each point of the curve. These data will form a regression curve that can be used with either linear (y=mx+b) or power (y=axb) fit analyses to interpolate the concentration of any unknown sample.
Compared to UV spectrophotometric methods, fluorescence demonstrates improved specificity because it is less affected by other components present in samples. As a result, fluorescent methods lead to a more accurate determination of nucleic acid concentration (Figure 5).
Real-Time PCR (qPCR)
Real-time PCR (also known as qPCR) is a technique that relies on thermal cycling consisting of repeated cycles of heating and cooling for DNA melting and enzymatic replication of targeted amplicons by DNA polymerase. After n rounds of thermal cycling, a total of 2n PCR products are formed. Detection instruments measure the accumulation of DNA product after each round of PCR amplification using fluorescent reporters. The reporters can be either dyes like SYBR® green or probes such as TaqMan®. RNA can be measured with the same process following a preliminary reverse transcription (RT) step converting it to cDNA.
The primary data from a real-time PCR experiment is an amplification curve, which shows the fluorescent signal in relative fluorescent units (RFUs) versus the cycle number and charts the accumulation of amplified product. The baseline is measured early in the amplification process before the instrument can detect product formation. As product accumulates, it reaches a point where the instrument is able to detect the change in signal above the background level–this is the exponential portion of the curve.
The detection threshold is the level of fluorescence where the product accumulation can be distinguished from the background. This threshold is automatically set above the baseline, within the exponential region of the amplification curve. The cycle number where the amplification product crosses that detection threshold is the Quantification Cycle (Cq) (Figure 6).
An unknown sample concentration can be estimated from its corresponding Cq number when compared to a standard curve of Cq values from samples of known concentration (Figure 7).
The real-time PCR strategy of quantitation offers several advantages over other methods:
- Real-time PCR offers the most sensitive detection, as low as picogram quantities of nucleic acid.
- Real-time PCR can accurately quantitate a subset of specific nucleic acids of interest, even in the presence of common contaminants, other nucleic acids, primers and free nucleotides. This is because PCR primers can be designed for sequence-specific targets (such as certain domains, species or genes) (Figures 8 and 9).
Because real-time PCR exploits the enzymatic replication of DNA polymerase, it only measures those molecules that are amplifiable. The implication is that highly degraded samples and fragments that won’t amplify in a downstream assay also won’t contribute to a concentration estimate.
|Table 1. The pros and cons of common quantitation methods.|
|UV Spectrophotometry||Quick, simple, does not require any reagents||Limited low-end sensitivity Inaccuracies introduced by contaminants Cannot discern between nucleic acid species|
|Fluorescence||Quick, simple Highly sensitive for low-level samples Contaminants pose minimal effect on results Preferential detection of nucleic acid species||Requires calibration with standard(s) Is not sequence-specific Does not measure amplifiability|
|Real-Time PCR||Sensitivity in picogram range Ability to design in sequence-specificity Measures amplifiability||Requires calibration with standards Requires costly reagents and advanced instrumentation Time-intensive assay setup with potential need for optimization work|