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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.


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
Denaturation95°C∼20 to 30 secondsDouble-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.
Primer Annealing48 to 72°C∼20 to 40 secondsAfter 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.
Extension68 to 72°C∼1 to 2 minutesAfter 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.

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.


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


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.


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 nm522 nm5x1 mL17591
Helixyte™ Green *10,000X Aqueous PCR Solution*498 nm522 nm1 mL17592
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*490 nm525 nm1 mL17597
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*490 nm525 nm10 mL17598
Q4ever™ Green *1250X DMSO Solution*503 nm527 nm100 µL17608
Q4ever™ Green *1250X DMSO Solution*503 nm527 nm2 mL17609


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.


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*3364551 g610
EDANS acid [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid] *CAS 50402-56-7*33645510 g611
EDANS C5 maleimide3364555 mg619
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] *CAS 100900-07-0*3364551 g615
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] *CAS 100900-07-0*33645510 g616
Tide Fluor™ 1 acid [TF1 acid] *Superior replacement for EDANS*341448100 mg2238
Tide Fluor™ 1 alkyne [TF1 alkyne]3414485 mg2237
Tide Fluor™ 1 amine [TF1 amine] *Superior replacement for EDANS*3414485 mg2239
Tide Fluor™ 1 azide [TF1 azide]3414485 mg2236
Tide Fluor™ 1 CPG [TF1 CPG] *500 Å*341448100 mg2240

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*454N/A5 g2001
DABCYL C2 amine454N/A100 mg2006
DABCYL C2 maleimide454N/A25 mg2008
DABCYL-DBCO454N/A5 mg2010
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4*454N/A1 g2004
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4*454N/A5 g2005
3'-DABCYL CPG *1000 Å*454N/A1 g6008
5'-DABCYL C6 Phosphoramidite454N/A1 g6009
Tide Quencher™ 1 acid [TQ1 acid]492N/A100 mg2190
Tide Quencher™ 1 alkyne [TQ1 alkyne]492N/A5 mg2189

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


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.


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
  • 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

 

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.
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
 

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*qPCR5786045 mL400
6-ROXtra™ fluorescence reference solution *25 uM for PCR reactions*qPCR5785955 mL398

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® 7700ROX6-FAM6-TET--
ABI PRISM® 7000 and 7900
Applied Biosystems® 7300
StepOnePlus™
ROX6-FAM6-TET6-HEX-
Applied Biosystems® 7500ROX6-FAM6-TET6-HEXTide Fluor™ 3
iFluor® 647
Alexa Fluor 647
Cy5

Table 9. Dye qPCR Calibration Plates For Real Time PCR

Product
Ex (nm)
Em (nm)
Unit Size
Cat No.
7 Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well* 1 Set67020
Cy3.5 Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5795911 Plate67014
FAM Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*4935171 Plate67006
JOE Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5205451 Plate67012
NED Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5455671 Plate67010
NED Dye qPCR Calibration Solution *10,000X*545567100 µL67031
ROX Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5786041 Plate67004
SYBR Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*4985221 Plate67008
TAMRA Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5525781 Plate67000
VIC Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5265431 Plate67002