Real-Time PCR (qPCR)

Real-time polymerase chain reaction, also referred to as quantitative PCR (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 a broad range of 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.

Figure 1. Like conventional PCR, qPCR utilizes the same three-step amplification procedure – denaturation, annealing, and extension (figure made in BioRender).

Table 1. Overview of the basic steps in the qPCR cycling reaction

Step	Temperature	Time	Process
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.
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. Note: It is critical to determine a proper temperature for the annealing step to ensure optimal efficiency and specificity. A typical annealing temperature is ~5°C below the melting temperature (Tm) of the primer.
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). Note: The required denaturation time may increase if template GC content is relatively high.

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 equipped 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 over the duration of the entire PCR run.

qPCR Fluorescence Detection Strategies

In qPCR, two fluorescent detection strategies are generally applied to measure amplicon concentration, dye-based and probe-based assays.

Dye-Based qPCR

Figure 2. qPCR using Helixyte™ Green (Cat No. 17591). 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).

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.

Figure 3. Quantitative PCR results targeting GAPDH with an input of 100 ng–0.00001 ng cDNA was 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.

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 to ensure amplification specificity, perform a melt curve analysis post-amplification.

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	5×1 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	1 mL	17598
Q4ever™ Green 2000X DMSO Solution	503 nm	527 nm	50 µL	17608
Q4ever™ Green 2000X DMSO Solution	503 nm	527 nm	1 mL	17609

Probe-Based qPCR

Figure 4. 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).

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, BXQ-1, BXQ-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.

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
Tide Fluor™ 1 CPG [TF1 CPG] 1000 Å	341	448	100 mg	2241
Tide Fluor™ 1 maleimide [TF1 maleimide] Superior replacement for EDANS	341	448	5 mg	2242
Tide Fluor™ 1 succinimidyl ester [TF1 SE] Superior replacement for EDANS	341	448	5 mg	2244
5(6)-FAM [5-(and-6)-Carboxyfluorescein] CAS 72088-94-9	493	517	1 g	100
5(6)-FAM [5-(and-6)-Carboxyfluorescein] CAS 72088-94-9	493	517	10 g	101
5(6)-FAM [5-(and-6)-Carboxyfluorescein] CAS 72088-94-9	493	517	25 g	102
5(6)-FAM cadaverine	493	517	100 mg	127
5(6)-FAM ethylenediamine	493	517	100 mg	123
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] CAS 117548-22-8	493	517	25 mg	110
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] CAS 117548-22-8	493	517	100 mg	111
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] CAS 117548-22-8	493	517	1 g	112
6-FAM [6-Carboxyfluorescein]	493	517	100 mg	106
6-FAM [6-Carboxyfluorescein]	493	517	1 g	107
6-FAM [6-Carboxyfluorescein]	493	517	5 g	108
6-FAM Alkyne	493	517	10 mg	134

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
Tide Quencher™ 1 amine [TQ1 amine]	492	N/A	5 mg	2192
Tide Quencher™ 1 azide [TQ1 azide]	492	N/A	5 mg	2188
Tide Quencher™ 1 CPG [TQ5 CPG] 500 Å	492	N/A	100 mg	2193
Tide Quencher™ 1 CPG [TQ5 CPG] 1000 Å	492	N/A	100 mg	2194
Tide Quencher™ 1 maleimide [TQ1 maleimide]	492	N/A	5 mg	2196
Tide Quencher™ 1 phosphoramidite [TQ1 phosphoramidite]	492	N/A	100 µmoles	2198
Tide Quencher™ 1 succinimidyl ester [TQ1 SE]	492	N/A	25 mg	2199
Tide Quencher™ 2 acid [TQ2 acid]	516	N/A	100 mg	2200
Tide Quencher™ 2 alkyne [TQ2 alkyne]	516	N/A	5 mg	2212
Tide Quencher™ 2 amine [TQ2 amine]	516	N/A	5 mg	2202
Tide Quencher™ 2 azide [TQ2 azide]	516	N/A	5 mg	2211
Tide Quencher™ 2 CPG [TQ5 CPG] 500 Å	516	N/A	100 mg	2203
Tide Quencher™ 2 CPG [TQ5 CPG] 1000 Å	516	N/A	100 mg	2204
Tide Quencher™ 2 phosphoramidite [TQ2 phosphoramidite]	516	N/A	100 µmoles	2208
Tide Quencher™ 2 succinimidyl ester [TQ2 SE]	516	N/A	25 mg	2210

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	–	–	–	–	+	+++	+	–
Cy5.5® Tide Fluor™ 6	–	–	–	–	–	+	+++	+
Cy7® Tide Fluor™ 7	–	–	–	–	–	–	+	+++

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.

figure-for-taquest-fast-qpcr-master-mix-with-helixyte-green-low-rox_TjrUP

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*.

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

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
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green	High Rox	1 mL	17280
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green	High Rox	5 mL	17281
TAQuest™ qPCR Master Mix for TaqMan Probes	No Rox	1 mL	17282
TAQuest™ qPCR Master Mix for TaqMan Probes	No Rox	5 mL	17283
TAQuest™ qPCR Master Mix for TaqMan Probes	Low Rox	1 mL	17284
TAQuest™ qPCR Master Mix for TaqMan Probes	Low Rox	5 mL	17285
TAQuest™ qPCR Master Mix for TaqMan Probes	High Rox	1 mL	17286
TAQuest™ qPCR Master Mix for TaqMan Probes	High Rox	5 mL	17287
TAQuest™ FAST qPCR Master Mix for TaqMan Probes	No Rox	1 mL	17288
TAQuest™ FAST qPCR Master Mix for TaqMan Probes	No Rox	5 mL	17289
TAQuest™ FAST qPCR Master Mix for TaqMan Probes	Low Rox	1 mL	17290
TAQuest™ FAST qPCR Master Mix for TaqMan Probes	Low Rox	5 mL	17291
TAQuest™ FAST qPCR Master Mix for TaqMan Probes	High Rox	1 mL	17292
TAQuest™ FAST qPCR Master Mix for TaqMan Probes	High Rox	5 mL	17293

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, and 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

Video 1. 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.

Table 7. Ordering ROX Reference Dyes

Product	Application	Ex (nm)	Em (nm)	Unit Size	Cat No.
ROX Reference Dye 50X fluorescence reference solution for PCR reactions	qPCR	578	604	5 mL	400
6-ROXtra™ fluorescence reference solution 25 uM for PCR reactions	qPCR	578	595	5 mL	398

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

Real-Time PCR (qPCR)

Basic Principles of qPCR

Table 1. Overview of the basic steps in the qPCR cycling reaction