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Intercalating Dyes or Fluorescent Probes For RT-qPCR?

The unique feature of real-time quantitative polymerase chain reaction (RT-qPCR) is that it associates the amplification of your target gene with a fluorescent signal in a quantifiable manner. Presently, there are numerous fluorescent tool kits/methods to consider when designing your RT-qPCR experiment. However, the two major categories to choose from are fluorescent intercalating dyes and fluorescent oligonucleotide probes. So which is best, dye or probe?

First Up: Intercalating Dyes (i.e. SYBR Green)

Intercalating fluorescent insert into DNA during the process of amplification, so the intensity of fluorescence increases respectively, and you can make measurements in real-time. Therefore, if you are amplifying your target samples for 40 cycles, the fluorescence detected at the end of the last cycle will be much higher compared to a measurement at cycle 10.

dsDNA + intercalating dye = measurable fluorescence

Therefore, since you have more DNA products at the end of the last cycle, your intensity of fluorescence also increases.

Pros

  • Very cost-effective when planning a large-scale experiment.
  • Saves time during the experimental design phase because it only requires the proper design of primers.

Cons

  • Not very specific. The dye intercalates into any dsDNA including primer dimers and non-specific products. Thus, careful analyses of results are needed.
    Hint: Always perform a melting curve analysis also known as a dissociation curve. This helps to ensure that you are only getting one product. It is very easy to do a melting curve, you just add the dissociation curve to your cycle by clicking the “add dissociation curve” option on the experimental setup. This produces the dissociation curve data along with the CT values of your samples. You can also check that the PCR product is only one band of the right size on a gel.
  • You can get less reliable results or skewing, if primer dimers or contaminating products are present. The CT value will be different. Again, you need a thorough analysis of the data.
  • Is more prone to producing non-specific fluorescence with targets that are in low abundance.
  • Requires more time for data analysis.

Next: Oligonucleotide Probes (i.e. Taqman)

As the name implies, this method involves the use of fluorescently labeled oligonucleotides (short DNA molecules). The probe is usually labeled both at the 5’ end and at the 3’ end. A fluorescent reporter is placed at the 5’ end of the probe and a fluorescent quencher at the 3’ end of the probe. As long as the reporter is in proximity to the quencher, no fluorescence will be detected. Thus, fluorescence is usually detected only after the 5’ end and the 3’ end of the fluorescently-labeled oligonucleotide probe separate. This usually occurs during the RT-qPCR reaction as a byproduct of the enzymatic activity of the polymerase when the probe incorporates into the PCR product.

Fluorescence detection using this method depends on two processes: 1) the binding of the primer to its target sequences 2) the binding of the probe to its complementary sequence downstream of the primer.

Pros

  • More likely to only amplify your desired product because of the combined specificity of your primers and probe.
  • No dissociation curve needed as fluorescence is only detected by the binding of the probe to the correct target.
  • Data is more reliable because of specificity.
  • Saves time during data analysis because of the specificity of the assay.

Cons

  • Not as cost-effective when planning large-scale experiments.
  • Takes longer to design the experiment because you need good primers and probes.

Overall, both fluorescence detection methods work great but one may work better for your specific experiment!

Further Reading

If you need more help deciding, check out this reference by Tajadini, M. et. al. (2014). Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res. 3:85. doi: 10.4103/2277-9175.127998

Best of luck!

Please comment below with your preference of dye or probe. Which would you rather?

Image credit: johnnyswatts

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