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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.

PCR workflow

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

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.

qPCR diagram

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.

Helixyte 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

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.

Probe-based 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.

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
Tide Fluor™ 1 CPG [TF1 CPG] *1000 Å*341448100 mg2241
Tide Fluor™ 1 maleimide [TF1 maleimide] *Superior replacement for EDANS*3414485 mg2242
Tide Fluor™ 1 succinimidyl ester [TF1 SE] *Superior replacement for EDANS*3414485 mg2244
5(6)-FAM [5-(and-6)-Carboxyfluorescein] *CAS 72088-94-9*4935171 g100
5(6)-FAM [5-(and-6)-Carboxyfluorescein] *CAS 72088-94-9*49351710 g101
5(6)-FAM [5-(and-6)-Carboxyfluorescein] *CAS 72088-94-9*49351725 g102
5(6)-FAM cadaverine493517100 mg127
5(6)-FAM ethylenediamine493517100 mg123
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*49351725 mg110
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*493517100 mg111
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*4935171 g112
6-FAM [6-Carboxyfluorescein]493517100 mg106
6-FAM [6-Carboxyfluorescein]4935171 g107
6-FAM [6-Carboxyfluorescein]4935175 g108
6-FAM Alkyne49351710 mg134
6-FAM Alkyne493517100 mg956
6-FAM Azide49351710 mg133
6-FAM Azide493517100 mg955
FAM-xtra™ Phosphoramidite 49351750 µmoles6037
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite]493517100 µmoles6016
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite]49351710x100 µmoles6017
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*49351710 mg116
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*493517100 mg117
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*4935171 g118
6-Fluorescein phosphoramidite498517100 µmoles6018
6-Fluorescein phosphoramidite49851710x100 µmoles6019
3'-(6-Fluorescein) CPG *1000 Å*4985171 g6014
Tide Fluor™ 2 acid [TF2 acid] *Superior replacement for fluorescein*50352525 mg2245
Tide Fluor™ 2 alkyne [TF2 alkyne] *Superior replacement for fluorescein*5035251 mg2253
Tide Fluor™ 2 amine [TF2 amine] *Superior replacement for fluorescein*5035251 mg2246
Tide Fluor™ 2 azide [TF2 azide] *Superior replacement for fluorescein*5035251 mg2252
Tide Fluor™ 2 maleimide [TF2 maleimide] *Superior replacement for fluorescein*5035251 mg2247
Tide Fluor™ 2, succinimidyl ester [TF2 SE] *Superior replacement for fluorescein*5035255 mg2248
Tide Fluor™ 2WS acid [TF2WS acid] *Superior replacement for FITC*50352510 mg2348
Tide Fluor™ 2WS amine [TF2WS amine] *Superior replacement for FITC*5035251 mg2351
Tide Fluor™ 2WS maleimide [TF2WS maleimide] *Superior replacement for FITC*5035251 mg2350
Tide Fluor™ 2WS succinimidyl ester [TF2WS SE] *Superior replacement for FITC*5035255 mg2349
6-TET alkyne5215435 mg245
6-TET azide5215435 mg244
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]52154350 µmoles6021
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]521543100 µmoles6027
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]52154310x100 µmoles6025
6-TET, SE [6-Carboxy-2',4,7',7-tetrachlorofluorescein, succinimidyl ester]5215435 mg211
Helix Fluor™ 545, succinimidyl ester5265431 mg250
VIC phosphoramidite52654350 µmoles6080
VIC phosphoramidite526543100 µmoles6081
VIC phosphoramidite5265431 g6082
5-VIC phosphoramidite52654350 µmoles6083
5-VIC phosphoramidite526543100 µmoles6084
5-VIC phosphoramidite5265431 g6085
6-VIC, SE [6-VIC NHS ester]5265431 mg212
6-VIC, SE [6-VIC NHS ester]5265435 mg213
6-HEX alkyne5335595 mg241
6-HEX azide5335595 mg240
6-HEX, SE [6-Carboxy-2',4,4',5',7,7'-hexachlorofluorescein, succinimidyl ester]5335595 mg202
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]533559100 µmoles6026
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]53355910x100 µmoles6024
6-NED alkyne5455671 mg216
6-NED azide5455671 mg217
6-NED maleimide5455671 mg218
6-NED, SE [6-NED NHS ester]5455671 mg214
6-NED, SE [6-NED NHS ester]5455671 mg215
Helix Fluor™ 575, succinimidyl ester5535701 mg251
Tide Fluor™ 3 acid [TF3 acid] *Superior replacement for Cy3*54657125 mg2268
Tide Fluor™ 3 alkyne [TF3 alkyne] *Superior replacement for Cy3*5465711 mg2255
Tide Fluor™ 3 amine [TF3 amine] *Superior replacement for Cy3*5465711 mg2269
Tide Fluor™ 3 azide [TF3 azide] *Superior replacement for Cy3*5465711 mg2254
Tide Fluor™ 3 maleimide [TF3 maleimide] *Superior replacement for Cy3*5465711 mg2270
Tide Fluor™ 3 succinimidyl ester [TF3 SE] *Superior replacement for Cy3*5465711 mg2271
Tide Fluor™ 3 phosphoramidite [TF3 CEP] *Superior replacement to Cy3 phosphoramidite*546571100 µmoles2274
Tide Fluor™ 3WS acid [TF3WS acid] *Superior replacement for Cy3*55156310 mg2268
Tide Fluor™ 3WS amine [TF3 amine] *Superior replacement for Cy3*5515631 mg2347
Tide Fluor™ 3WS maleimide [TF3 maleimide] *Superior replacement for Cy3*5515631 mg2344
Tide Fluor™ 3WS succinimidyl ester [TF3WS SE] *Superior replacement for Cy3*5515631 mg2346
Tide Fluor™ 4 acid [TF4 acid] *Superior replacement for ROX and Texas Red*57860210 mg2285
Tide Fluor™ 4 alkyne [TF4 alkyne] *Superior replacement for ROX and Texas Red*5786021 mg2301
Tide Fluor™ 4 amine [TF4 amine] *Superior replacement for ROX and Texas Red*5786021 mg2286
Tide Fluor™ 4 azide [TF4 azide] *Superior replacement for ROX and Texas Red*5786021 mg2300
Tide Fluor™ 4 maleimide [TF4 maleimide] *Superior replacement for ROX and Texas Red*5786021 mg2287
Tide Fluor™ 4, succinimidyl ester [TF4 SE] *Superior replacement for ROX and Texas Red*5786025 mg2289
Tide Fluor™ 5WS acid [TF5WS acid] *Superior replacement for Cy5*64966410 mg2278
Tide Fluor™ 5WS alkyne [TF5WS alkyne] *Superior replacement for Cy5*6496641 mg2276
Tide Fluor™ 5WS amine [TF5WS amine] *Superior replacement for Cy5*6496641 mg2279
Tide Fluor™ 5WS azide [TF5WS azide] *Superior replacement for Cy5*6496641 mg2275
Tide Fluor™ 5WS maleimide [TF5WS maleimide] *Superior replacement for Cy5*6496641 mg2280
Tide Fluor™ 5WS succinimidyl ester [TF5WS SE] *Superior replacement for Cy5*6496645 mg2281
Tide Fluor™ 6WS acid [TF6WS acid] *Superior replacement for Cy5.5*68270110 mg2291
Tide Fluor™ 6WS alkyne [TF6WS alkyne] *Superior replacement for Cy5.5*6827011 mg2303
Tide Fluor™ 6WS amine [TF6WS amine] *Superior replacement for Cy5.5*6827011 mg2292
Tide Fluor™ 6WS azide [TF6WS azide] *Superior replacement for Cy5.5*6827011 mg2302
Tide Fluor™ 6WS maleimide [TF6WS maleimide] *Superior replacement for Cy5.5*6827011 mg2293
Tide Fluor™ 6WS succinimidyl ester [TF6WS SE] *Superior replacement for Cy5.5*6827011 mg2294
Tide Fluor™ 7WS acid [TF7WS acid] *Superior replacement for Cy7*75678010 mg2330
Tide Fluor™ 7WS alkyne [TF7WS alkyne] *Superior replacement for Cy7*7567801 mg2305
Tide Fluor™ 7WS amine [TF7WS amine] *Superior replacement for Cy7*7567801 mg2331
Tide Fluor™ 7WS azide [TF7WS azide] *Superior replacement for Cy7*7567801 mg2304
Tide Fluor™ 7WS maleimide [TF7WS maleimide] *Superior replacement for Cy7*7567801 mg2332
Tide Fluor™ 7WS succinimidyl ester [TF7WS SE] *Superior replacement for Cy7*7567801 mg2333
Tide Fluor™ 8WS acid [TF8WS acid] *Near Infrared Emission*78580110 mg2335
Tide Fluor™ 8WS alkyne [TF8WS alkyne] *Near Infrared Emission*7858011 mg2307
Tide Fluor™ 8WS amine [TF8WS amine] *Near Infrared Emission*7858011 mg2336
Tide Fluor™ 8WS azide [TF8WS azide] *Near Infrared Emission*7858011 mg2306
Tide Fluor™ 8WS maleimide [TF8WS maleimide] *Near Infrared Emission*7858011 mg2337
Tide Fluor™ 8WS succinimidyl ester [TF8WS SE] *Near Infrared Emission*7858011 mg2338

Table 4. Quencher dyes for labeling the 5' end or 3' end on sequence-specific qPCR probes.

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
Tide Quencher™ 1 amine [TQ1 amine]492N/A5 mg2192
Tide Quencher™ 1 azide [TQ1 azide]492N/A5 mg2188
Tide Quencher™ 1 CPG [TQ5 CPG] *500 Å*492N/A100 mg2193
Tide Quencher™ 1 CPG [TQ5 CPG] *1000 Å*492N/A100 mg2194
Tide Quencher™ 1 maleimide [TQ1 maleimide]492N/A5 mg2196
Tide Quencher™ 1 phosphoramidite [TQ1 phosphoramidite]492N/A100 µmoles2198
Tide Quencher™ 1 succinimidyl ester [TQ1 SE]492N/A25 mg2199
Tide Quencher™ 2 acid [TQ2 acid]516N/A100 mg2200
Tide Quencher™ 2 alkyne [TQ2 alkyne]516N/A5 mg2212
Tide Quencher™ 2 amine [TQ2 amine]516N/A5 mg2202
Tide Quencher™ 2 azide [TQ2 azide]516N/A5 mg2211
Tide Quencher™ 2 CPG [TQ5 CPG] *500 Å*516N/A100 mg2203
Tide Quencher™ 2 CPG [TQ5 CPG] *1000 Å*516N/A100 mg2204
Tide Quencher™ 2 phosphoramidite [TQ2 phosphoramidite]516N/A100 µmoles2208
Tide Quencher™ 2 succinimidyl ester [TQ2 SE]516N/A25 mg2210
Tide Quencher™ 2WS acid [TQ2WS acid]516N/A25 mg2050
Tide Quencher™ 2WS alkyne [TQ2WS alkyne]516N/A1 mg2213
Tide Quencher™ 2WS alkyne [TQ2WS alkyne]516N/A5 mg2214
Tide Quencher™ 2WS maleimide [TQ2WS maleimide]516N/A1 mg2059
Tide Quencher™ 2WS succinimidyl ester [TQ2WS, SE]516N/A5 mg2058
BXQ-1 Alkyne522N/A1 mg2414
BXQ-1 Amine522N/A5 mg2406
BXQ-1 Azide522N/A1 mg2412
BXQ-1 Carboxylic Acid522N/A25 mg2400
BXQ-1 CPG (500 Å)522N/A100 mg2408
BXQ-1 CPG (1000 Å)522N/A100 mg2410
BXQ-1 Maleimide522N/A1 mg2404
BXQ-1 Succinimidyl Ester522N/A5 mg2402
5-TAMRA [5-Carboxytetramethylrhodamine] *CAS 91809-66-4*55257810 mg363
5-TAMRA [5-Carboxytetramethylrhodamine] *CAS 91809-66-4*552578100 mg364
5-TAMRA [5-Carboxytetramethylrhodamine] *CAS 91809-66-4*5525781 g365
Rhodamine aldehyde [5-TAMRA aldehyde]5525785 mg9005
5-TAMRA alkyne5525785 mg487
5-TAMRA alkyne55257850 mg961
5-TAMRA azide5525785 mg486
5-TAMRA azide55257850 mg960
5-TAMRA cadaverine5525785 mg356
5-TAMRA ethylenediamine5525785 mg358
5-TAMRA C6 maleimide5525785 mg424
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-68-7*5525785 mg373
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-68-7*552578100 mg374
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-68-7*5525781 g375
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] *CAS 98181-63-6*552578100 mg360
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] *CAS 98181-63-6*5525781 g361
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] *CAS 98181-63-6*5525785 g362
5(6)-TAMRA cadaverine55257825 mg355
5(6)-TAMRA ethylenediamine55257825 mg354
5(6)-TAMRA Maleimide [Tetramethylrhodamine-5-(and-6)-maleimide]5525785 mg412
5(6)-TAMRA C6 maleimide5525785 mg423
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] *CAS 246256-50-8*55257825 mg370
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] *CAS 246256-50-8*552578100 mg371
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] *CAS 246256-50-8*5525781 g372
6-TAMRA [6-Carboxytetramethylrhodamine] *CAS 91809-67-5*55257810 mg366
6-TAMRA [6-Carboxytetramethylrhodamine] *CAS 91809-67-5*552578100 mg367
6-TAMRA [6-Carboxytetramethylrhodamine] *CAS 91809-67-5*5525781 g368
6-TAMRA alkyne5525785 mg491
6-TAMRA alkyne55257850 mg966
6-TAMRA azide5525785 mg490
6-TAMRA azide55257850 mg965
6-TAMRA cadaverine5525785 mg357
6-TAMRA CPG *1000 Å*5525781 g6051
6-TAMRA ethylenediamine5525785 mg359
6-TAMRA Maleimide [Tetramethylrhodamine-6-maleimide] *CAS 174568-68-4*5525781 mg419
6-TAMRA C6 maleimide5525785 mg425
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-69-8*5525785 mg376
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-69-8*552578100 mg377
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-69-8*5525781 g378
BXQ-2 Alkyne554N/A1 mg2434
BXQ-2 Amine554N/A5 mg2426
BXQ-2 Azide554N/A1 mg2432
BXQ-2 Carboxylic Acid554N/A25 mg2420
BXQ-2 CPG (500 Å)554N/A100 mg2428
BXQ-2 CPG (1000 Å)554N/A100 mg2430
BXQ-2 Maleimide554N/A1 mg2424
BXQ-2 Succinimidyl Ester554N/A5 mg2422
Tide Quencher™ 3 acid [TQ3 acid]573N/A100 mg2220
Tide Quencher™ 3 alkyne [TQ3 alkyne]573N/A5 mg2232
Tide Quencher™ 3 amine [TQ3 amine]573N/A5 mg2222
Tide Quencher™ 3 azide [TQ3 azide]573N/A5 mg2231
Tide Quencher™ 3 CPG [TQ5 CPG] *500 Å*573N/A100 mg2223
Tide Quencher™ 3 CPG [TQ5 CPG] *1000 Å*573N/A100 mg2224
Tide Quencher™ 3 maleimide [TQ3 maleimide]573N/A5 mg2226
Tide Quencher™ 3 phosphoramidite [TQ3 phosphoramidite]573N/A100 µmoles2228
Tide Quencher™ 3 succinimidyl ester [TQ3 SE]573N/A25 mg2230
Tide Quencher™ 3WS acid [TQ3WS acid]573N/A1 mg2229
Tide Quencher™ 3WS succinimidyl ester [TQ3WS SE]573N/A5 mg2227
Tide Quencher™ 4 CPG [TQ5 CPG] *500 Å*603N/A100 mg2062
Tide Quencher™ 4 CPG [TQ5 CPG] *1000 Å*603N/A100 mg2063
Tide Quencher™ 4WS acid [TQ4WS acid]603N/A5 mg2060
Tide Quencher™ 4WS alkyne [TQ4WS alkyne]603N/A1 mg2069
Tide Quencher™ 4WS amine [TQ4WS amine]603N/A1 mg2061
Tide Quencher™ 4WS azide [TQ4WS azide]603N/A1 mg2068
Tide Quencher™ 4WS maleimide [TQ4WS maleimide]603N/A1 mg2064
Tide Quencher™ 4WS succinimidyl ester [TQ4WS SE]603N/A1 mg2067
Tide Quencher™ 5 CPG [TQ5 CPG] *500 Å*661N/A100 mg2077
Tide Quencher™ 5 CPG [TQ5 CPG] *1000 Å*661N/A100 mg2078
Tide Quencher™ 5WS acid [TQ5WS acid]661N/A5 mg2075
Tide Quencher™ 5WS alkyne [TQ5WS alkyne]661N/A1 mg2083
Tide Quencher™ 5WS amine [TQ5WS amine]661N/A1 mg2076
Tide Quencher™ 5WS azide [TQ5WS azide]661N/A1 mg2082
Tide Quencher™ 5WS maleimide [TQ5WS maleimide]661N/A1 mg2079
Tide Quencher™ 5WS succinimidyl ester [TQ5WS SE]661N/A1 mg2081
Tide Quencher™ 6WS acid [TQ6WS acid]694N/A5 mg2090
Tide Quencher™ 6WS alkyne [TQ6WS alkyne]694N/A1 mg2098
Tide Quencher™ 6WS amine [TQ6WS amine]694N/A1 mg2091
Tide Quencher™ 6WS azide [TQ6WS azide]694N/A1 mg2097
Tide Quencher™ 6WS maleimide [TQ6WS maleimide]694N/A1 mg2094
Tide Quencher™ 6WS succinimidyl ester [TQ6WS SE]694N/A1 mg2096
Tide Quencher™ 7WS acid [TQ7WS acid]764N/A5 mg2105
Tide Quencher™ 7WS alkyne [TQ7WS alkyne]764N/A1 mg2113
Tide Quencher™ 7WS amine [TQ7WS amine]764N/A1 mg2106
Tide Quencher™ 7WS azide [TQ7WS azide]764N/A1 mg2112
Tide Quencher™ 7WS maleimide [TQ7WS maleimide]764N/A1 mg2109
Tide Quencher™ 7WS succinimidyl ester [TQ7WS SE]764N/A1 mg2111

Table 5. Recommended FRET pairs for developing FRET oligonucleotides

Donor \ Acceptor
Tide Fluor™ 1+++++++-----
Tide Fluor™ 2
Tide Fluor™ 3
Texas Red®
Tide Fluor™ 4---+++++--
Tide Fluor™ 5
Tide Fluor™ 6
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.

Amplification Plot

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™ GreenTAQuest™ qPCR Master Mix for Probe-based Detection
PrincipleUses 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 formatOptimized for qPCR and 2-step RT-qPCROptimized for qPCR and 2-step RT-qPCR
Detection sensitivityVariable1-10 copies
Gene expressionLow level of quantitationHigh level of quantitation
  • 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
  • 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
  • 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.

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, 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

ROX Comparitive Analysis
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

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.

Reference Dye
Reporter Dye 1
Reporter Dye 2
Reporter Dye 3
Reporter Dye 4
ABI PRISM® 7000 and 7900
Applied Biosystems® 7300
Applied Biosystems® 7500ROX6-FAM6-TET6-HEXTide Fluor™ 3
iFluor® 647
Alexa Fluor 647

Table 9. Dye qPCR Calibration Plates For Real Time PCR

Ex (nm)
Em (nm)
Unit Size
Cat No.
ReadiView™ Blue Real-Time PCR Visualization Dye *200X*500 Tests17300
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
ROX Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5786041 Plate67004
FAM Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*4935171 Plate67006
SYBR Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*4985221 Plate67008
NED Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5455671 Plate67010
JOE Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5205451 Plate67012
Cy3.5 Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*5795911 Plate67014
Cy5 Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well*6516701 Plate67016
7 Dye qPCR Calibration Plate *Optimized for ABI7500 Fast 96-Well* 1 Set67020
VIC Dye qPCR Calibration Solution *10,000X*526543100 µL67030
NED Dye qPCR Calibration Solution *10,000X*545567100 µL67031
FAM Dye qPCR Calibration Solution *10,000X*493517100 µL67032
Cy5 Dye qPCR Calibration Solution *10,000X*651670100 µL67033
ROX Dye qPCR Calibration Solution *10,000X*578595100 µl67034