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).
Table 2. End-point RT-PCR detection methods For Quantifying RT-PCR products
Advantages, disadvantages and applications of end-point RT-PCR
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 4. 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 |
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 5 below) and a non-fluorescent quencher dye at the 3′ end (see Table 6 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.
We offer a broad range of dye phosphoramidites and dye CPG supports for developing FRET oligonucleotides, TaqMan® probes and Molecular Beacons. This set includes classic dyes such as FAM, HEX, TET and JOE, as well as superior oligo-labeling dyes such as Tide Fluor™ and Tide Quencher™ dyes.
Table 5. 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 |
Tide Fluor™ Dyes for Labeling Oligos and Peptides
Tide Fluor™ dyes are a series of donor dyes optimized for developing FRET oligonucleotides and peptides for a variety of biological applications. Compared to common donor dyes such as EDANS, FAM, TAMRA, ROX, Cy 3 and Cy5, Tide Fluor™ dyes exhibit stronger fluorescence and higher photostability. They are the best affordable fluorescent dyes for labeling peptides and oligonucleotides without sacrificing performance.
Table 8. Tide Fluor™ Dyes and Spectral Properties For Quantifying RT-PCR products