While PCR technology has been around since the mid-1980s, it wasn’t until the 1990s that the tools and protocols became available to make PCR a truly quantitative method. Since then, quantitative, real-time PCR (often abbreviated as qPCR) has developed into one of the primary tools for measuring gene expression, microRNAs, copy number variance, and other applications.
Figure 1. PCR amplification stages. The exponential phase favors the true doubling of the target and provides the most accurate quantitation. It is during this phase that qPCR measurements are taken. During the linear phase, decreased reaction components slow the amplification, and the target is no longer doubling. At the plateau, the amplification has stopped. It is this stage that more qualitative PCR (or endpoint) measurements are taken.
The PCR amplification reaction has three distinct phases (Figure 1). With qPCR, the measurements are taken in the exponential phase when conditions favor the target doubling. The exponential phase provides the best conditions for accurate quantification because none of the components are in limiting supply, which can cause bias in the reaction.
Both qPCR and reverse transcription qPCR (RT-qPCR) (Figure 2) have gained in popularity due to the wide availability of instrumentation and low cost per data point. These techniques are more sensitive than earlier methods for gene expression and quantification such as northern blotting, and are more amenable to high-throughput analyses in part because of the standardization of labware and the availability of laboratory robotics for automated liquid handling.
Figure 2. qPCR and RT-qPCR workflow comparison. RT-qPCR requires that the starting RNA sample be reverse transcribed into a complementary single-stranded DNA prior to amplification steps. Two approaches to incorporating this additional step exist: 1-step RT-qPCR and 2 step RT-PCR.
Whether you are performing qPCR (DNA template) or RT-qPCR (RNA template), the quality of the starting material is very important because of the sensitivity of the reactions and the reaction components. First, DNA and RNA isolation methods should limit the potential for shearing and nicking of the nucleic acid, and in the case of RNA, ensure the removal of genomic DNA sequences. Precautions to avoid nuclease contamination, such as the use of nuclease inhibitors during purification, wearing gloves, and using nuclease-free labware, should be taken, especially when isolating and using RNA. After the nucleic acid samples are isolated, sample integrity can be easily evaluated by agarose gel electrophoresis. For RNA, the MIQE guidelines also recommend using microfluidic tools to determine the RNA integrity number (RIN) for all RNA samples.1 A larger RIN is indicative of intact RNA samples that will perform well in subsequent RT-qPCR reactions.
Numerous qPCR and RT-qPCR reaction inhibitors and contaminants can co-purify with the DNA or RNA samples. With homebrew procedures, trace amounts of phenol, chelators, and Proteinase K, which can inhibit thermostable polymerases, may be present in the final sample. RNA samples may contain small amounts of contaminants such as detergents, chelators, guanidine salts, phosphate, or pyrophosphate, all of which affect the reverse transcriptases used in the initial step to produce single-stranded cDNA. Likewise the contaminants from a particular source material may also impact the reactions. For example, samples extracted from soil may contain the strong inhibitor humic acid, whereas samples prepared from plants may contain polysaccharide contaminants. While many researchers rely on commercially available DNA and RNA isolation kits, which typically produce samples of adequate purity, there is still a chance that contaminants may slip through. For optimal isolation, nucleic acid extraction kits specifically validated for use with a specific sample or source are recommended.2
Prior to setting up qPCR reactions, the DNA or RNA samples should be analyzed by UV spectrophotometry or by the newer, more sensitive fluorometric readers to determine both quantity and possible purity of the samples (Table 1). However, these results still may not totally rule out the presence of certain contaminants or genomic DNA contamination. In order to comply with MIQE guidelines, it is necessary to include multiple control reactions that will help determine the presence of reaction inhibitors or genomic DNA contamination, such as no template control, environmental control, negative control of extraction, positive control, positive control of extraction, and standard curves, among others. While these additional controls will create a more complex experimental setup, they also help to eliminate misleading results that can influence further research efforts.
Table 1. UV spectrophotometric targets for DNA and RNA quantity and quality
Initially, qPCR reactions were performed in individual tubes; however, as experimental design became more complex with increased samples and controls, the need for higher throughput became more important. The reactions moved to multi-well plates (e.g., 96- and 384-well plates) with smaller reaction volumes, smaller wells, and more densely packed reactions. This shift in experimental design created issues with reaction setup that could lead to possible cross-contamination and the addition of erroneous amounts of reaction components. To alleviate these risks, researchers use master mixes that contain all reaction components except the template. Many researchers have also turned to automated liquid handlers, such PIPETMAX® with qPCR Assistant, to minimize errors arising from manually pipetting many different reactions.3,4
Master mixes (without enzyme and primers) that are optimized for a manufacturer’s specific DNA polymerase are available commercially. While these products offer a high level of convenience, preformulated master mixes limit the flexibility in optimizing an experiment. For example, thermostable DNA polymerases require Mg2+. Too much or too little Mg2+ can lead to the presence of nonspecific PCR products or inefficient reactions with lower PCR yields, respectively. To gain flexibility in reaction conditions, many researchers prepare their own master mixes for qPCR reactions by combining individual reagents, using the supplier’s reaction buffer minus Mg2+ and dNTPs as the base and optimizing Mg2+ as well as other components such as primers. Numerous variations of master mixes may be required on a given plate because individual qPCR reactions might require different primer combinations or fluorescent labels. This arrangement can lead to errors in mixing and addition of components, which leads many scientists to use a system such as PIPETMAX® with qPCR Assistant. Such a system insures correct preparation of master mixes and provides a record of the manipulations performed for reporting purposes.
Once master mixes are ready, the DNA or RNA sample(s) are added at a unified (normalized) concentration calculated based on the UV or fluorescence measurements as described above. The MIQE guidelines recommend normalization of RNA samples across all reactions, another tedious and error-prone task. Using a system like PIPETMAX® with Normalization Assistant minimizes these issues.
The techniques of qPCR and RT-qPCR have grown in popularity because of the analytical power they offer; however, there are numerous steps and components that can lead to errors or failure to obtain the results sought. Keep the following in mind to address these potential risks before and during the reaction setup and achieve verifiable results throughout:
1. Bustin, SA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55(4):611-22. (2009).
2. Kovac, K, et al. Improved data reproducibility using MIQE-compliant, automated qPCR workflows. In: MIQE & qPCR iBook: How to apply the MIQE guidelines – a visual, interactive and practical qPCR guide. Nour, AAA and Pfaffl, MW (eds). Apple iBook, 2015. ISBN 9783000488061.)
3. Bratz M, and Hook, B. Precise Evaluation of Plant RNA Extraction Methods with Automated RT-qPCR Assay Preparations. www.Gilson.com, Application Note, 2014.
4. Gluckenberger, DJ, et al. A combined fabrication and instrumentation platform for sample preparation. J. Lab. Automation. 19(3) 267-274 (2014).