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Reverse Transcription PCR (RT-PCR)

Reverse transcription polymerase chain reaction (RT-PCR) is a core molecular technique designed to detect and quantitate total RNA or messenger RNA (mRNA), with a high degree of sensitivity and accuracy. In this modified version of the standard PCR process, mRNA, which serves as the initial template, is first reversed transcribed to complementary DNA (cDNA) and then subsequently amplified via PCR for downstream analysis. Relative to other techniques for measuring mRNA, such as Northern blot analysis, RNAse protection assays, or in situ hybridization, RT-PCR is significantly more robust at detecting the RNA transcript of any gene regardless of its relative abundance. Consequently, RT-PCR is widely used to quantitatively study gene expression, examine transcript variants, and generate cDNA templates for cloning and sequencing. Furthermore, RT-PCR has become an instrumental diagnostic tool for the detection of pathogens, including viruses that cause Ebola, HIV and the novel coronavirus disease (Covid-19).



Principles of Reverse Transcription PCR

In order to apply PCR to the study of RNA, the RNA sample must first undergo the process of reverse transcription to generate cDNA, using the enzyme reverse transcriptase. This RNA-dependent DNA polymerase, with the assistance of reverse transcription primers, deoxynucleotides (dNTPs: dATP, dTTP, dGTP and dCTP) and enzyme cofactors, catalyzes the synthesis of cDNA from its respective target RNA sequence. The resulting cDNA is then used as the template for PCR amplification, and depending upon the manner in which reverse transcription and PCR are executed this process can be classified as either one-step RT-PCR or two-step RT-PCR.

Video 1. RT-PCR animation. Animated video tutorial illustrating the key steps in reverse transcription polymerase chain reaction: (1) reverse transcription, (2) denaturation, (3) primer annealing, (4) primer extension.


Table 1. Deoxynucleotides (dNTPs) for use in PCR, real-time PCR, and reverse transcription PCR

Unit Size
Cat No.
ReadiUse™ dNTP Mix *10 mM*Water5 mL17200
ReadiUse™ ddNTP Terminator Mix *10 mM*Water100 nmoles17205
ReadiUse™ dNTP Mix Set *10 mM PCR Grade*Water1 mL17258
ReadiScript™ RT Reverse Transcription Kit 50 Reactions60100


One-Step RT-PCR

In one-step RT-PCR, reverse transcription (i.e. cDNA synthesis) and PCR are carried out in the same reaction vessel and buffer. This simple and convenient single-tube design works well when performing a small number of assays and is amenable to high-speed, high-throughput applications. In one-step RT-PCR, sequence-specific primers are required to direct both the synthesis and amplification of cDNA. Since specific primers more readily anneal at higher reaction temperatures than random primers, it is best to utilize reverse transcriptases with high thermal stability when using this approach.

One step RT-PCR workflow

One-step RT-PCR diagram. In one-step RT-PCR, cDNA synthesis via reverse transcription (RT) and subsequent PCR amplification occur in the same reaction vessel (figure made in BioRender).

While the one-step RT-PCR approach offers several advantages, it is not without its caveats. Since both reverse transcription and PCR take place in the same tube the reaction conditions cannot be separately optimized, which can impact yield or reaction efficiency to varying degrees. Moreover, because all the cDNA synthesized is consumed in the subsequent PCR step, additional aliquots of the original RNA sample(s) are required in order to repeat reactions or to assess the expression of other genes.

Advantages, disadvantages and applications for one-step RT-PCR

One-Step RT-PCR
RT Primers
  • Gene-specific primers
  • Quick setup and minimal sample handling
  • Single closed-tube reaction eliminates the possibility of contamination between reverse transcription and PCR
  • Fewer pipetting manipulations minimizes potential errors
  • Easy processing of multiple samples for repetitive tests, or high-throughput screening
  • Improved data reproducibility
  • Must repeat or save RNA aliquot and perform new RT to analyze new target or repeat amplifications
  • Reaction conditions are not optimal—efficiency and thus quantification are affected
  • Primer dimers a bigger potential problem
  • Less sensitive than two-step because the reaction conditions are a compromise between the two combined reactions
  • Detection of fewer targets per sample
Ideal Applications
  • Working with multiple RNA samples to amplify only a few targets
  • High-throughput applications
  • Performing assays repeatedly


Two-Step RT-PCR

In two-step RT-PCR, reverse transcription and PCR are carried out in separate reaction vessels with different buffers, reaction conditions and priming strategies. Rather than employing only gene-specific primers like the one-step method, two-step RT-PCR is carried out using a mixture of random hexamers, oligo-dT primers, and/or gene-specific primers, which provides a full cDNA archive of all the RNA species in the sample. Furthermore, the uncoupling of reverse transcription and PCR allows for each reaction to be optimized individually for maximum efficiency and sequence representation. This permits a higher yield of cDNA during reverse transcription and provides an opportunity for cDNA samples to be stored for future amplification reactions or downstream applications.

Two step RT-PCR workflow

Two-step RT-PCR diagram. In two-step RT-PCR, cDNA synthesis via reverse transcription (RT) and subsequent PCR amplification occur in separate reaction vessels (figure made in BioRender).

While two-step RT-PCR is highly sensitive it is significantly more time-consuming and less amenable to high-speed, high-throughput application than one-step RT-PCR. It requires an additional open-tube step, more pipetting and sample handling, which can increase variability and the risk of contamination.

Advantages, disadvantages and applications for two-step RT-PCR

Two-Step RT-PCR
RT Primers
  • Oligo(dT) primers
  • Random hexamer primers
  • Gene-specific primers
  • A mixture of all three
  • Choice of RT primers
  • Flexible reaction optimization for maximum yield
  • Ability to perform multiple PCR reactions on the same cDNA sample
  • Greater flexibility to select RT enzymes and DNA polymerases for PCR separately
  • Ability to store cDNA for future use
  • Ideal method for applications with limited starting materials
  • Requires more setup, hands-on, and machine time
  • Additional open-tube step and pipetting increases the chances for experimental errors and contamination
  • Uses more reagents
  • Requires more optimization than one-step
Ideal Applications
  • Amplifying multiple targets from a single RNA source
  • If you plan to store cDNA for additional amplifications or future reuse


Measuring RT-PCR Amplicons

There are two primary approaches for measuring amplicon generation in RT-PCR. The first approach analyzes amplicon amplification after all the cycles of PCR have been completed and is referred to as end-point RT-PCR. The second approach measures amplicon concentration in real-time as it occurs throughout the PCR cycling process and is known as real-time RT-PCR or quantitative RT-PCR (RT-qPCR). In RT-qPCR, several different types of chemistries can be used to detect amplicon generation in real-time, these include Helixyte™ Green, Molecular Beacons and TaqMan probes.

End-point RT-PCR

End-point RT-PCR analysis, which is based on the plateau phase of PCR reaction, is used to analyze amplified products after all the cycles of the PCR reaction have been completed. In this method, amplicons are separated by agarose gel electrophoresis and visualized using a DNA binding dye, such as ethidium bromide (EtBr), to determine the size of the DNA molecules in the range of 500 to 30,000 bp. While EtBr is the most commonly used dye for visualizing DNA, it is mutagenic and highly toxic through inhalation. Instead, consider using safer and more environmentally friendly, non-toxic alternatives such as Gelite™ X100 (Cat No. 17706), Helixyte™ Green (Cat No. 17590), Helixyte™ Gold (Cat No. 17595), Gelite™ Green (Cat No. 17589) or Gelite™ Orange (Cat No. 17594).

Advantages, disadvantages and applications of end-point RT-PCR

End-Point RT-PCR
  • Convenient - suitable for analysis of any double-strand DNA sequence
  • Economical - cheaper than using custom designed probes
  • Poor precision and low sensitivity
  • Short dynamic range < 2 logs
  • Low resolution, size-based discrimination only
  • Not suitable for automation
  • Results are not expressed as numbers
  • Post-PCR processing
  • Gene expression analysis
  • Cloning
  • Contructing cDNA libraries
  • RNA sequencing
  • Microarray probe development
  • Species identification

Table 2. Nucleic acid stains for agarose and polyacrylamide gel electrophoresis

Ex (nm)¹
Unit Size
Cat No.
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution*254 mnLong path green filter1 mL17590
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution*254 mnLong path green filter100 µL17604
Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution*254 mnLong path green filter1 mL17595
Gelite™ Green Nucleic Acid Gel Staining Kit254 nm or 300 nmLong path green filter1 Kit17589
Gelite™ Orange Nucleic Acid Gel Staining Kit254 nm or 300 nmLong path green filter1 Kit17594
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters100 µL17700
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters500 µL17701
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters1 mL17702
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters10 mL17703
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters100 µL17704
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters500 µL17705
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters1 mL17706
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters10 mL17707

Quantitative RT-PCR

In quantitative RT-PCR (RT-qPCR), fluorescent DNA-intercalating dyes or sequence-specific fluorescent probes are integrated into the RT-PCR reaction allowing for amplicon concentration to be measured in real-time during the exponential phase of PCR. By combining amplification and detection into a single-step, RT-qPCR provides greater precision and accuracy and produces quantitative data with a dynamic range several orders of magnitude larger than end-point RT-PCR. Because of its higher sensitivity, RT-qPCR is routinely used to analyze mRNA in gene expression, to examine the presence of retroviruses and to validate results obtained by array analyses.

Helixyte™ Green for RT-qPCR

RT-qPCR using fluorescent DNA-intercalating dyes, such as Helixyte™ Green (Cat No.17591) provides the easiest and most economical method for detecting and quantitating PCR amplicons in real-time. Helixyte™ Green binds to double-stranded DNA (dsDNA), and when excited emits light. As amplicon concentration increases with each successive cycle of amplification, so does the fluorescence intensity of Helixyte™ Green, to a degree proportional to the amount of dsDNA present in each PCR cycle. Helixyte™ Green is a much safer alternative than the highly mutagenic EtBr and can be used to monitor the amplification of any dsDNA sequence with greater sensitivity and less PCR inhibition. Because DNA-intercalating dyes will bind to any dsDNA, such as primer-dimers and non-specific products, it is important to use well-designed primers to avoid amplifying non-target sequences. To ensure amplification specificity and to check for primer-dimer artifacts, a melt curve analysis should be performed post-amplification.

Table 3. 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

TaqMan® Probes and Molecular Beacons for RT-qPCR

In probe-based RT-qPCR, fluorescently-labeled, target-specific probes are used to measure DNA amplification in real-time. This method benefits from extreme specificity and affords the end-user the opportunity for multiplexing multiple targets in a single reaction. Of the many probe-based RT-qPCR chemistries available, TaqMan® probes and Molecular Beacons, are the most widely used and both depend upon Förster Resonance Energy Transfer (FRET) to generate a fluorescence signal. TaqMan® probes rely on the 5'-nuclease activity of Taq DNA polymerase. Short oligonucleotide sequences, complementary to the target of interest, are labeled with a fluorescent reporter dye at the 5' end (see Table 4 below) and a non-fluorescent quencher dye at the 3' end (see Table 5 below). During PCR cycling, primers and probe anneal to the target. As Taq DNA polymerase binds to and extends the primer upstream of the probe, the hybridized probe 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.

Like TaqMan® probes, Molecular Beacons are labeled with a fluorescent reporter dye at the 5' end and a non-fluorescent quencher dye at the 3' end. However, this method does not rely on the 5' nuclease activity of Taq DNA polymerase to generate a signal, rather Molecular Beacons are designed to remain intact during the entire amplification process. In the absence of the target, Molecular Beacons remain in a 'hairpin' confirmation due to its self-complementary stem structure. This brings both the fluorescent reporter and quencher dyes within close proximity of one another preventing the probe from fluorescing. When the Molecular Beacon hybridizes to its target, the fluorescent reporter and the quencher are separated, and the reporter dye emits at its characteristic wavelength.

Table 4. 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 5. 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 6. 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