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.
Table of Contents
Basic Principles of qPCR
Like conventional PCR, qPCR utilizes the same three-step amplification procedure - denaturation, annealing, and extension (figure made in BioRender).
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.
Table 1. Overview of the basic steps in the qPCR cycling reaction
Step ▲ ▼ | Temperature ▲ ▼ | Time ▲ ▼ | Process ▲ ▼ |
Denaturation | 95°C | ∼20 to 30 seconds | Double-stranded DNA (dsDNA) template is heated to high temperature. This disrupts the hydrogen bonds between the complementary base pairs causing dsDNA to separate into single-stranded DNA (ssDNA).
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Primer Annealing | 48 to 72°C | ∼20 to 40 seconds | After denaturation, the reaction temperature is lowered to ∼48 to 72°C. This promotes the binding of forward and reverse primers to each of the ssDNA templates and the subsequent binding of DNA polymerases to the primer-template hybrid.
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Extension | 68 to 72°C | ∼1 to 2 minutes | After annealing, the reaction temperature is raised to ∼68 to 72°C. This enables DNA polymerase to extend the primers, synthesizing new DNA strands complementary to the ssDNA template in the 5’ to 3’ direction. |
qPCR Fluorescence Detection Strategies
Two detection strategies are generally applied to measure amplicon concentration in qPCR, dye-based and probe-based chemistries.
DNA polymerase extends the sequence-specific primer during the extension phase by incorporating dNTPs complementary to the DNA template. As newly synthesized double-stranded DNA is produced, Helixyte™ Green; will bind to the DNA complexes and fluoresce (figure made in BioRender).
Dye-Based qPCR
Quantitative PCR results targeting GAPDH with an input of 100 ng-0.00001 ng cDNA were performed using Helixyte™ Green *20X Aqueous PCR Solution* (Cat No. 17591) and a Fast Advanced Master Mix on an Applied Biosystems® 7500 FAST Real-Time PCR System.
Table 2. Double-stranded DNA-binding dyes for qPCR
Product ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
Helixyte™ Green *20X Aqueous PCR Solution* | 498 nm | 522 nm | 5x1 mL | 17591 |
Helixyte™ Green *10,000X Aqueous PCR Solution* | 498 nm | 522 nm | 1 mL | 17592 |
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution* | 490 nm | 525 nm | 1 mL | 17597 |
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution* | 490 nm | 525 nm | 10 mL | 17598 |
Q4ever™ Green *1250X DMSO Solution* | 503 nm | 527 nm | 100 µL | 17608 |
Q4ever™ Green *1250X DMSO Solution* | 503 nm | 527 nm | 2 mL | 17609 |
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.
Illustration of probe-based qPCR. As DNA polymerase extends the primer during elongation, it hydrolyzes sequence-specific probes that have annealed to the single-stranded DNA template, separating the reporter dye from the quencher and resulting in an amplification-dependent increase in fluorescence (figure made in BioRender).
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.
Table 3. Fluorescent reporter dyes for labeling the 5' end or 3' end on sequence-specific qPCR probes.
Product ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
EDANS acid [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid] *CAS 50402-56-7* | 336 | 455 | 1 g | 610 |
EDANS acid [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid] *CAS 50402-56-7* | 336 | 455 | 10 g | 611 |
EDANS C5 maleimide | 336 | 455 | 5 mg | 619 |
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] *CAS 100900-07-0* | 336 | 455 | 1 g | 615 |
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] *CAS 100900-07-0* | 336 | 455 | 10 g | 616 |
Tide Fluor™ 1 acid [TF1 acid] *Superior replacement for EDANS* | 341 | 448 | 100 mg | 2238 |
Tide Fluor™ 1 alkyne [TF1 alkyne] | 341 | 448 | 5 mg | 2237 |
Tide Fluor™ 1 amine [TF1 amine] *Superior replacement for EDANS* | 341 | 448 | 5 mg | 2239 |
Tide Fluor™ 1 azide [TF1 azide] | 341 | 448 | 5 mg | 2236 |
Tide Fluor™ 1 CPG [TF1 CPG] *500 Å* | 341 | 448 | 100 mg | 2240 |
Table 4. Quencher dyes for labeling the 5' end or 3' end on sequence-specific qPCR probes.
Product ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
DABCYL acid [4-((4-(Dimethylamino)phenyl)azo)benzoic acid] *CAS 6268-49-1* | 454 | N/A | 5 g | 2001 |
DABCYL C2 amine | 454 | N/A | 100 mg | 2006 |
DABCYL C2 maleimide | 454 | N/A | 25 mg | 2008 |
DABCYL-DBCO | 454 | N/A | 5 mg | 2010 |
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4* | 454 | N/A | 1 g | 2004 |
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4* | 454 | N/A | 5 g | 2005 |
3'-DABCYL CPG *1000 Å* | 454 | N/A | 1 g | 6008 |
5'-DABCYL C6 Phosphoramidite | 454 | N/A | 1 g | 6009 |
Tide Quencher™ 1 acid [TQ1 acid] | 492 | N/A | 100 mg | 2190 |
Tide Quencher™ 1 alkyne [TQ1 alkyne] | 492 | N/A | 5 mg | 2189 |
Table 5. Recommended FRET pairs for developing FRET oligonucleotides
Donor \ Acceptor ▲ ▼ | DABCYL ▲ ▼ | TQ1 ▲ ▼ | TQ2 ▲ ▼ | TQ3 ▲ ▼ | TQ4 ▲ ▼ | TQ5 ▲ ▼ | TQ6 ▲ ▼ | TQ7 ▲ ▼ |
EDANS | +++ | +++ | + | - | - | - | - | - |
MCA | +++ | +++ | + | - | - | - | - | - |
Tide Fluor™ 1 | +++ | +++ | + | - | - | - | - | - |
FAM FITC | + | + | +++ | + | - | - | - | - |
Cy2® Tide Fluor™ 2 | + | + | +++ | + | - | - | - | - |
HEX JOE TET | - | - | + | +++ | + | - | - | - |
Cy3® TAMRA Tide Fluor™ 3 | - | - | + | +++ | + | - | - | - |
ROX Texas Red® | - | - | - | + | +++ | + | - | - |
Tide Fluor™ 4 | - | - | - | + | +++ | + | - | - |
Cy5® Tide Fluor™ 5 | - | - | - | - | + | +++ | + | - |
TAQuest™ qPCR Master Mixes
Amplification plot for a dilution series of HeLa cells cDNA amplified in replicate reactions to detect GAPDH using TAQuest™ FAST qPCR Master Mix with Helixyte™ Green *Low ROX*.
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 |
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Advantages |
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Disadvantages |
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Table 6. TAQuest™ qPCR Master Mixes.
Product ▲ ▼ | Reference Dye ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
TAQuest™ qPCR Master Mix with Helixyte™ Green | No Rox | 1 mL | 17270 |
TAQuest™ qPCR Master Mix with Helixyte™ Green | No Rox | 5 mL | 17271 |
TAQuest™ qPCR Master Mix with Helixyte™ Green | Low Rox | 1 mL | 17272 |
TAQuest™ qPCR Master Mix with Helixyte™ Green | Low Rox | 5 mL | 17273 |
TAQuest™ qPCR Master Mix with Helixyte™ Green | High Rox | 1 mL | 17274 |
TAQuest™ qPCR Master Mix with Helixyte™ Green | High Rox | 5 mL | 17275 |
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green | No Rox | 1 mL | 17276 |
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green | No Rox | 5 mL | 17277 |
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green | Low Rox | 1 mL | 17278 |
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green | Low Rox | 5 mL | 17279 |
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
Stability comparison of PCR reference solutions (6-ROX, 6-ROXtra™, and 6-ROX analog). The blue bar represents starting fluorescence. The red bar represents the remaining fluorescence after 4 weeks at 25°C.
- 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
Table 8. Possible ROX Reference and reporter dye combinations for multiplex qPCR assays.
Instrument ▲ ▼ | Reference Dye ▲ ▼ | Reporter Dye 1 ▲ ▼ | Reporter Dye 2 ▲ ▼ | Reporter Dye 3 ▲ ▼ | Reporter Dye 4 ▲ ▼ |
ABI PRISM® 7700 | ROX | 6-FAM | 6-TET | - | - |
ABI PRISM® 7000 and 7900 Applied Biosystems® 7300 StepOnePlus™ | ROX | 6-FAM | 6-TET | 6-HEX | - |
Applied Biosystems® 7500 | ROX | 6-FAM | 6-TET | 6-HEX | Tide Fluor™ 3 iFluor® 647 Alexa Fluor 647 Cy5 |