Is It more cost effective to analyze multiple genes using TaqMan versus traditional qRT-PCR? What other main advantages and disadvantages exist for each method?
I’m trying to analyze several genes specific to bone cells. I’m interested in specifically looking at: OCN, ALP, RUNX2, Sox9, TRAP, CatK, VEGF, CD34, and COL1A.
Hello Genesis Marrero
It is more cost-effective to use the traditional qRT-PCR, especially if you are assaying several different genes. But the disadvantage of qRT-PCR is the use of SYBR Green, a dsDNA binding dye, which is used to quantify amplicon amount during the course of PCR. SYBR Green dye binds primer dimers and non-specific products as well as specific products (because it cannot discriminate between different double-stranded DNAs). The SYBR Green method can give you false positive results and this incorrect and shifted data can finally lead to decreased performance. In such a case, you may have to perform the melting curve analysis to check reaction specificity.
On the other hand, TaqMan assay, though expensive offers two significant advantages namely, 1) the TaqMan assay only measures amplification progression of the target sequence, as the probes are target specific, and 2) you can monitor the quantity of various qPCR products in a single reaction by adding different primers and TaqMan probes with different reporter dyes to the master mix. This multiplex approach allows you to detect several fluorescent signals at the end of every thermal cycle. Thus, TaqMan-based PCR will give you high specificity and sensitivity (low copy number) and high reproducibility.
I would suggest you use TaqMan assay to analyze several genes if your budget permits since TaqMan probes are expensive.
Good Luck!
Depending on the throughput of samples it might not be worth optimizing all these assays (and identify a set of suitable reference genes). It might be easier and eventually cheaper to go for RNA-seq, even if only very little of the RNA-seq output will be used.
If using qrtPCR, I would not recommend TaqMan if you don't go for multiplexing (what comes with its own difficulties). The higher detection specificity of TaqMan probes as compared to dsDNA-specific dyes like SYBR Green makes it more difficult to confirm specific amplification what is important to ensure the interpretability of Ct values. If you use TaqMan probes you will need to run post-PCR gels to check what all was amplified.
Another scenario except multiplexing where using TaqMan probes would be advantagous is if you would not go for quantification but only for detection, possibly of single molecules, and if the amplification of the assay would not be sufficiently specific (e.g. amplifying primer-dimers).
Taqman (or similar probe assay) allows you to set up multiplex reactions. Not only you can assess more than one target in the same samples in the same well, you can easily use a housekeeping gene as internal control for normalization to compare among samples that may have different amount of input RNA.
I also have agree with Jochen's statements. If you are using it for relative quantitation, you'd have to make sure the probes are not competing with each other, what if one takes over resources from the other and amplifies better as a result which is what Jochen was hinting at I think (qualitative analysis). It sounds nice on paper being able to multiplex several targets, but in reality it also has some potential caveats. I wouldn't necessarily say that Taqman is more specific, SYBR-green or intercalating dye-based methods can be just as specific as long as the primers are specific.
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