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Designing a TaqMan Gene Expression Assay
TaqMan assay design is an important step in the process of quantifying gene expression with significant specificity and sensitivity. TaqMan assays utilize fluorescent probes to detect and quantify specific nucleic acid sequences, offering a robust and reliable method for real-time PCR (qPCR) research applications. The accuracy of these assays makes them valuable for a wide range of research areas, including gene expression analysis, and biomarker discovery. However, the success of a TaqMan gene expression assay depends on careful design and optimization. This involves selecting appropriate target sequences, designing effective primers and probes, and verifying assay performance to support accuracy and reproducibility. This guide will provide an overview of the essential principles and steps involved in designing a TaqMan gene expression assay, equipping researchers with the knowledge to develop assays that yield reliable and meaningful data.
Thermo Fisher Scientific design pipeline
Thermo Fisher Scientific uses an innovative oligonucleotide probe/primer design pipeline. This is accomplished by developing robust primer design algorithms and an extensive array of bioinformatics tools and processes to automate assay design. The pipeline also integrates design details with the manufacturing process and assay quality control (QC).
Although the predesigned TaqMan gene expression assays are extensively used as determined by their frequent citations in scientific journal articles, some researchers still need to design their own assays. If a predesigned assay is not available, consider using the Custom Assay Design Tool to take advantage of the proprietary design algorithm and rigorous bioinformatic interrogations. Leverage our proprietary Bioinformatics algorithms to design your own customized assay using the Assay Design Hub.
Below is a discussion of considerations to have when you design your own assay.
Evaluation of target sequences to help ensure specificity
 | Oligonucleotide probe and primer (assay) design is a important element in the experimental design process for real-time qPCR experiments. When designing an assay, consider the following bioinformatics criteria that should be addressed for the successful design of a primer/probe set. |
- The key feature in assay design is specificity of the assay for the transcript of interest. It is important that the fluorescent signal being detected during real time PCR is specific to the target transcript, and that there is no contribution from homologous sequences that might complicate the interpretation of the quantitative data. High target specificity is assured by comparing the sequence of the designed probe and primers to other sequences in the transcriptome from which the gene is transcribed. Check sequence uniqueness with a BLAST® database search. The transcript specificity problem can be difficult to tackle because of high homology between closely related genes, alternative splicing within a single gene, and the potential presence of transcribed pseudogenes. We have been able to design robust assays to deal with many of these specificity issues using our bioinformatics design pipeline and continue to refine our design algorithms to tackle the challenging design problems.
- For gene expression assays, it is also important that the assay is specific for detecting cDNA and not gDNA (genomic DNA). RNA samples can often be contaminated with significant amounts of gDNA, depending on the RNA purification methods utilized. The genomic DNA contamination problem can be solved with high-quality RNA purification chemistries, use of highly active DNase, and rigorous QC of the prepared RNA samples. During the qPCR experiment it is recommended to run a “No RT” (Reverse Transcriptase) control to determine if any of the signal is a result of gDNA contamination. Designing primers across exon-exon boundaries also helps avoid gDNA amplification. If possible, place the probe, rather than one of the primers, over the exon-exon boundary so that the primers bind in two distinct exons. Placing the probe over the exon-exon boundary helps ensure that the fluorescent signal is only generated from templates that have correctly spliced exons.
- An assay should be designed in a region of unambiguous sequence that does not contain any known single nucleotide polymorphisms (SNPs) or repeat sequences.
TaqMan assay design considerations
Once a unique “location” on the transcript of interest has been decided, the next step in creating your own TaqMan gene expression assay is to design primers and a probe.
 | When designing the assay, put the forward and reverse primers as close as possible to the probe while avoiding overlap. Amplicons should be kept short to help ensure doubling at each PCR cycle. Amplicons of 50–150 base pairs are recommended to promote efficient amplification and sensitivity of qPCR. The amplicon should span an exon-exon boundary to prevent amplification of target gene in gDNA. |
 | Once a unique “location” on the transcript of interest has been decided, the next step in creating your own TaqMan gene expression assay is to design primers and a probe. GC content is an important consideration when designing primers. Here, the last five nucleotides on the 3’ end should have no more than two G and/or C nucleotides, as this could lead to non-specific product formation. The optimal primer length is 20 bases and Tm should be kept at 58–60°C (10°C lower than that of the probe, enabling the use of universal thermal cycling parameters). For both primers and probe, keep GC content at 30–80%, and avoid runs of four or more G nucleotides for efficient amplification. The primers should be specific to the target gene and, if you synthesized several primer sets, you should always select the pair of primers that generates the highest signal-to-noise ratio (with no amplification of genomic DNA in the case of gene expression assays). You can do this using our Primer Express software. This will help you to design primers and probes based on the sequence of interest, while considering important parameters, such as melting temperature (Tm) and GC content. The software is optimized for use with TaqMan reagents and universal thermal cycling conditions. Default variables have already been standardized, thus streamlining the process. |
 | For many TaqMan gene expression assays, when DNA or cDNA is used as the template, a concentration of 900 nM for primers and 250 nM for the probe should enable a highly sensitive assay. |
Additional tools and guidelines for assay design
 | If designing a primer/probe assay set is not where you want to spend your time (and hope you get it right) order a predesigned TaqMan Gene Expression Assay or let us design one for you. Use the Custom Assay Design Tool for designing custom TaqMan assays and select the Custom Plus option, which offers bioinformatics analysis of target sequences, in-silico QC step, and specificity for either gene- or transcript-level detection. If you do not have an input sequence, the assay design tool will help you to search for sequences by gene symbol or location in the genome, e.g., across a range of model species. Once designed, you can reorder a custom assay at your own convenience. All assay sequences remain entirely confidential. |
Using proprietary algorithms, the Custom Plus option performs thorough checks on your sequences—such as optimal Tm requirements, GC content, buffer and salt conditions, oligonucleotide concentrations, secondary structure formation and amplicon size—so the assay is successful. The in-silico QC pipeline also removes designs that are not highly specific to the gene of interest or that might detect homologous genes or pseudogenes.
Thermo Fisher Scientific’s assay design guidelines enable a reliable procedure for designing your own assay and must be followed completely for the optimal results.
Through our extensive experience with probe and primer design of TaqMan assays for quantitative RT-PCR, we have empirically determined the parameters useful for selecting oligonucleotide sequences that are most likely to result in successful, functional assays. Before designing your own assay, we recommend that you check whether there is a predesigned assay that meets your needs using our comprehensive Assay Search Tool. Our aim is to provide the quantitative assays that will fit the requirements of the entire spectrum of sample types and sample preparation methodologies utilized by the broad range of users of a particular assay.
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