Polymerase chain reaction (PCR) and reverse transcription quantitative real-time PCR (RT-qPCR) are fundamental molecular techniques that have become widespread across many laboratories worldwide owing to their sensitive detection of a specific DNA sequence or a specific RNA transcript or viral genome sequence. Once we understand how these techniques detect their nucleic acid targets, their distinct roles become clear in terms of the instruments and reagents to use.
A PCR assay can be performed in one of two ways, either using endpoint PCR, where the PCR reaction goes to completion before final fluorescence is measured, or with real-time PCR detection, in which fluorescence is measured as it occurs (in ‘real-time’) during the PCR amplification process. When endpoint PCR is used, a simple thermocycler can suffice for the PCR amplification and detection. If real-time PCR is required, then the assay must be performed on a real-time thermocycler instrument, as it has the appropriate detection format for the assay and can also detect a known RNA transcript or sequence in RNA.
In this overview, we will focus on key considerations and approaches to enable a RT-qPCR method, since this newer technique offers a level of sensitive and specific detection for very popular applications like gene expression and molecular and genotyping analysis.
Instrument features play an important role in qPCR detection results and vary across offerings. Thermal stability, optical systems, and throughput format also influence your experimental setup and process.
A system’s thermal stability across the entire plate during the amplification process ensures that you can run your assay in all wells of the plate, including the exterior wells around the plate. Not having to worry about edge effects during PCR amplification makes the whole process more efficient with less pipetting, leading to reduced waste.
To interrogate the reaction, real-time thermocyclers must illuminate the sample and detect the resultant fluorescence. For consistent intensity and pathlength, it is important to ensure that consistent illumination and detection occur across all wells. In some instances, this can be achieved by having LED/detector pairs mounted on a shuttle that scans across a plate. Other optic configurations, such as using fixed LEDs to illuminate an entire plate, using multiple LEDs in a shuttle, or the use of fiber optics to position the light from a fixed LED, could result in edge or angle effects, variation in LED intensity, or varying pathlength.
Depending on assay design and available sample quantities, the choice of different well formats may be an important consideration. Offerings from 96-, 96 deep-, and 384-well are available, accommodating different starting sample volumes for varying application needs.
In addition to instrument selection, a robust qPCR process includes assay design. It will be important to consider how to design each step of a qPCR assay, from the sample preparation to the assay design itself.
Assay design includes consideration around single-plex assays versus multiplex assays for several targets. Multiplex assays are more complex to design and optimize, even if you are purchasing a commercial assay, but they offer great cost and throughput advantages if you’ll be using them often. However, in multiplex PCR, it’s important to have a robust master mix for the qPCR reaction that allows for amplification of each target, reducing contamination potential and sample-to-sample variability.
qPCR detection for the presence of amplified DNA can be measured by two methods: dye- or probe-based approaches. In the ideal situation, as DNA is amplified, the number of amplicons present in a reaction—proportional to the log of the initial number of targets—will double with each cycle. Fluorescence intensity, being proportional to the quantity of amplicons, doubles with it.
When intercalating dyes, such as SYBR and EvaGreen, bind to the nucleic acids, they will cause the nucleic acid to become fluorescent, and therefore detectable. They are inexpensive and require a relatively simple assay design for optimizing qPCR reactions. Because the dyes themselves are not sequence-specific, and will bind to any double-stranded DNA, it’s necessary to run a melting point curve analysis to assure the assay has amplified only a single product.
Fluorescently labeled probes, such as TaqMan, rely on a sequence-specific probe bound to a specific target region of interest, resulting in fluorescence. In comparison to intercalating dyes, probe-based assays are more costly and complex to design, however, they offer a specificity that allows them to interrogate targets that are very similar to other sequences. They can also be multiplexed allowing for multiple target detection within a single reaction.
The reagents necessary for a reaction are often sold as supermixes, which contain a polymerase, dNTPs, cations, buffer, and other additives commonly used in PCR reactions. The polymerases are enzymes essential for DNA replication creating identical copies of DNA strands from the original DNA template.
A standard DNA polymerase—already significantly more advanced than the original Taq—is adequate for most qPCR applications. But for difficult-to-amplify samples—such as those with high GC content, a large concentration of inhibitors in the matrix, secondary structure, or long amplicons—it may be best to use an advanced, highly robust, highly processive, polymerase engineered for purpose.
Preparing a master mix from the supermix allows for a consistent reagent mix, saving time while also reducing pipetting, contamination potential, and sample-to-sample variability. It’s important to use a mix that is specifically intended either for dye-based or probed-based chemistry, as applicable.
In general, running technical replicates and averaging Cq values will allow for better reproducibility in sampling technique. This can be seen as beneficial in the case of the following:
Taking note of these key considerations around instrument selection and assay planning will allow for the most reliable data from your qPCR assay, saving you time and additional effort.