Behind the Scenes: Designing TaqMan Assays – Ask TaqMan #39

If you work with different species or conduct transgenic experiments, you might ask a question like Manuel in Toronto –

He asked: “Some of your pre-designed TaqMan gene expression assays offer species specific options, e.g. human specific with no cross-reactivity with mouse. Is this something I can request to be made available?

Well Manuel, to answer your question, it is worth looking at what exactly goes into designing a TaqMan Assay. There are over 2 million pre-designed TaqMan Gene Expression Assays, covering more than 28 different species and each assay has gone through a robust design process to enable a reliable, consistent, and high performing TaqMan assay.

The whole process begins with good transcript sequences from the NCBI Reference Sequence Project database, a public source for highly curated, non-redundant transcripts.

Once we have a starting transcript- repetitive and low complexity regions are masked, along with all single nucleotide polymorphisms. This ensures the primer and probe designs avoid these regions.

Exon-exon boundaries are also mapped as target sequences.  When possible, TaqMan probes are designed across exon-exon boundaries so that the primers will bind in two distinct exons. This ensures that the fluorescent signal is only generated from templates that have correctly spliced exons.

These preprocessed transcripts are then passed through a proprietary program for assay design. This automated process uses a set of algorithms, based on our knowledge and expertise in assay design, that will result in successful, functional assays

This includes parameters such as optimal Tm requirements, GC content, buffer and salt conditions, oligo concentrations, secondary structure, optimal amplicon size, and reduction of primer-dimer formation.

Once a design is ready, it is assessed by an in silico QC pipeline, which scores each assay design. This is done to screen out designs that are not highly specific to the gene of interest or that might detect homologous genes or pseudogenes.

The assay design with the best score from QC testing is chosen for manufacturing as a TaqMan Gene Expression Assay. The design is added to our website and becomes available for order. This entire design process happens in the background for every assay to enable the best possible design.

All 2 million gene expression assays have been designed to be specific to the target transcripts in the species it was designed for, but are not cross checked against other species during the design process.  Since many of you like Manuel also want to check our assays against other species, we created a transgenic cross-reactivity (see screenshot) check feature available on our online assay search tool.  You’ll see it on the search results page.

When searching for an assay, check the box for the species that you do not want the assay to detect.  In this example, we are looking for a human assay that will not detect mouse sequences.  All the assays that remain, will not detect mouse.

Example of FOXP3 Search in TaqMan gene expression assay tool:

In the search results, the assay labeled “best coverage” is the recommended product based on the filtered criteria selected.  You can also view related publications related to the assay target in the starred area if you wish to expand your research.

It’s also worth checking out our other great resources on the importance of TaqMan assay design.