Real-Time PCR (qPCR)

Real-time polymerase chain reaction (qPCR) is a highly sensitive technique routinely used in molecular biology to detect and quantify specific oligonucleotide sequences or analyze variations in gene expression levels. By combining both the DNA amplification and detection process in a single step, qPCR enables researchers to measure relative or absolute amplicon concentration in real-time, as it is being generated throughout the PCR cycling process. Apart from gene expression studies, qPCR has found utility in many molecular biology applications, including genotyping, drug target validation, biomarker discovery, pathogen detection, and measuring RNA interference. Furthermore, with minor modifications to the basic protocol and the addition of the enzyme reverse transcriptase, qPCR can be adapted to monitor mRNA levels to detect and diagnose viral infections.

Basic Principles of qPCR

---

In qPCR, the same amplification procedure is used as in conventional PCR. A segment of target DNA, which serves as a template, is combined in a single tube along with other components essential to the amplification reaction (e.g., thermostable DNA polymerases, forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and reaction buffer). The prepared tube is placed in an instrument, referred to as a thermal cycler. A thermal cycler is a laboratory apparatus that typically has a thermal block with holes where tubes holding the PCR reaction mixture are inserted. The reaction contents are then subjected to a series of time and temperature-dependent steps - denaturation, primer annealing, and extension (see Table 1) - This series of steps is repeated 25 to 30 times, resulting in an exponential amplification of the DNA template.



The major differences between qPCR and conventional PCR are two-fold. First, qPCR provides the opportunity to integrate various fluorescent detection strategies, such as using DNA-intercalating dyes or sequence-specific fluorescent probes, to measure amplicon concentration after each PCR cycle. Fluorescence is monitored throughout the entire PCR process, and the amount of fluorescence generated during amplification is directly proportional to the amount of amplified DNA produced. Second, qPCR requires a thermal cycler with an optical detection module to measure the fluorescence signal generated. By plotting fluorescence intensity versus the cycle number, qPCR instruments can generate curves known as amplification plots, representing the accumulation of amplicons throughout the entire PCR run.

qPCR Fluorescence Detection Strategies

---

Two detection strategies are generally applied to measure amplicon concentration in qPCR, dye-based and probe-based chemistries.

In dye-based qPCR assays, DNA-intercalating fluorophores, such as Helixyte™ Green bind specifically to the minor groove of dsDNA to measure DNA amplification as it occurs throughout the PCR process. Alone such dyes display weak background fluorescence, but fluorescence intensity is enhanced significantly upon binding to dsDNA. As amplicon concentration increases with each successive cycle of amplification, so does the fluorescence intensity of the dye, to a degree proportional to the amount of dsDNA present in each PCR cycle. Set-up for this type of qPCR reaction is simple and convenient. All the necessary components, including two sequence-specific primers, a DNA template, and the DNA-binding dye, are mixed in a single reaction tube. This allows for the amplification and detection of PCR products to occur simultaneously and eliminates the need for any post-PCR manipulations.

Dye-Based qPCR

Compared to microarrays, dye-based qPCR is more sensitive at detecting modest changes in expression levels, making it well-suited for investigating small subsets of genes. Although dsDNA-binding dyes provide the most convenient and cost-effective option for qPCR, the principal drawback to intercalation-based detection is that it is not sequence-specific. Any dsDNA produced from off-target and non-template amplification (NTC) will be observed, resulting in less accurate quantification. To check for primer-dimer artifacts and ensure amplification specificity, perform a melt curve analysis post-amplification.

Probe-Based qPCR

In probe-based qPCR, sequence-specific fluorescent probes are used in combination with primers to detect the amplification product. Of the many probe-based qPCR chemistries available, including hybridization probes and molecular beacons, the most widely used employs the 5' nuclease assay associated with Taq DNA polymerase.

Probes for 5' nuclease assays are synthesized with a fluorescent reporter dye (see Table 3 below), such as FAM, HEX, NED, TET, VIC, Cy3, or Tide Fluor™ dyes, covalently attached to the 5' end and a quencher dye (see Table 4 below), such as DABCYL, TAMRA, AzoDye-1, AzoDye-2 or Tide Quencher™ dyes, to the 3' end of a short oligonucleotide, which is complementary to the target DNA sequence. While the probe is intact, the reporter and quencher remain in close proximity to each other, FRET occurs, and consequently, the reporter dye signal is quenched. During PCR cycling, both the primers and probe anneal to the target. As Taq DNA polymerase binds to and extends the primer upstream of the probe, any probe bound to the correct target sequence is hydrolyzed, and the fragment containing the reporter dye is released. The fluorescence signal can now be detected, and the amount of fluorescence signal generated is proportional to the amount of qPCR products produced. See Table 5 below for information on fluorescent reporter/quencher pairs commonly used in qPCR.



Not only does this method benefit from high sensitivity and specificity, but it also allows multiplexing using probes with different combinations of reporter dyes. This allows for an increase in throughput, meaning multiple samples can be assayed per plate, and consequently, there is a reduction in both sample and reagent usage.

TAQuest™ qPCR Master Mixes

---

The TAQuest™ qPCR Master Mixes are 2X concentrated, ready-to-use mixes designed to simplify the reaction assembly for qPCR without compromising sensitivity, specificity, or PCR efficiency. In a convenient pre-mixed solution, TAQuest™ qPCR Master Mixes contain all the essential components needed to perform a qPCR experiment, except for the template, primers, and probes (if using). It includes a TAQuest™ Hot Start Taq DNA Polymerase enzyme to facilitate reaction set up at room temperature, PCR-grade dNTPs, MgCl₂, as well as enhancers and stabilizers in an optimized reaction buffer. Some TAQuest™ qPCR Master Mixes also include Helixyte™ Green, a double-stranded DNA binding dye for detecting PCR products during amplification, and a ROX passive reference dye to assist with troubleshooting and increase data precision.



The AAT Bioquest portfolio includes master mixes for two different real-time PCR detection chemistries, Helixyte™ green and probe-based (e.g., TaqMan). Helixyte™ green TAQuest™ qPCR Master Mixes include the dsDNA binding dye Helixyte™ green in the reaction mixture to directly detect target amplification via nonspecific binding to dsDNA. Helixyte™ green master mixes are cost-effective and a flexible option when using target species not pre-defined. Probe-based TAQuest™ qPCR Master Mixes provide better specificity and are compatible with running multiplex assays. These master mixes are ready-to-use cocktails containing most of the reagents for amplifying and detecting DNA in qPCR; only the template, primers, and probe need to be added to the reaction mixture.

Summary of TAQuest™ qPCR Master Mixes for dye- and probe-based detection chemistries.

	TAQuest™ qPCR Master Mix with Helixyte™ Green	TAQuest™ qPCR Master Mix for Probe-based Detection
Principle	Uses Helixyte™ green, a double-stranded DNA binding dye used to detect amplicons as it accumulates during PCR.	It uses a fluorogenic probe specific to the target gene to detect amplicons as it accumulates during PCR.
qPCR format	Optimized for qPCR and 2-step RT-qPCR	Optimized for qPCR and 2-step RT-qPCR
Specificity	Medium	High
Detection sensitivity	Variable	1-10 copies
Reproducibility	Medium	High
Multiplexing	No	Yes
Gene expression	Low level of quantitation	High level of quantitation
Applications	Gene expression DNA quantitation Pathogen detection CHiP	Gene expression DNA quantitation Pathogen detection CHiP SNP genotyping Copy number variation Pathway analysis microRNA & small RNAs Mutation detection Protein analysis
Advantages	It can be used to monitor any dsDNA sequence No probe is required, making assay set up easier and more cost-effective	High specificity, sensitivity, and reproducibility due to specific hybridization between probe and target Compatible with multiplexing Eliminates post-PCR processing
Disadvantages	Helixyte™ green can bind to nonspecific dsDNA sequences, increasing the risk of false-positive signals Imperative that primers are well-designed to minimize amplification of non-target sequences	Each sequence requires design and optimization for different probes Predesigned probes can be costly to purchase

ROX Reference Dye for Real-Time PCR

---

Passive reference dyes, such as the ROX reference dye, are essential to the accuracy and reproducibility of qPCR reactions performed on ROX-dependent PCR cyclers (e.g., the Applied Biosystems^® instruments). When added to the qPCR master mix, the ROX Reference Dye is designed to normalize the fluorescent signal of qPCR reporter dyes or hybridization probes and account for non-PCR-related fluorescence signal variations, including pipetting errors, bubbles, condensation, evaporative loss, and instrument variation. Since the ROX Reference Dye does not interfere with the qPCR reaction and has an easily discernable red emission spectrum, it provides an excellent baseline in multiplex qPCR assays. Although ROX has been widely used in qPCR, its convenience is limited by its low stability. Moreover, ROX must be cold-stored to preserve its fluorescence.

6-ROXtra™ Fluorescence Reference Solution

AAT Bioquest^® 6-ROXtra™ - with spectral properties nearly identical to ROX - has considerable stability and water solubility improvements. Compared to ROX, 6-ROXtra™ offers a convenient method for normalizing fluorescent reporter signals in qPCR without modifying the instrument's default analysis parameters. In addition, 6-ROXtra™ can act as an internal control to improve the precision of qPCR data by:

• Normalizing for non-PCR related fluorescence signal variations due to uneven illumination, pipetting inaccuracies, sample effects, bubbling in the wells or well position
• Normalizing for fluorescence fluctuations (e.g., machine noise)
• Creating a stable baseline in multiplex qPCR assays