a variety of TaqMan assay product packaging

The accuracy and reliability of quantitative PCR (qPCR) experiments is of paramount importance in molecular biology. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines provide a standardized framework for the execution and reporting of qPCR assays, aimed at achieving reproducibility and credibility of experimental results. Compliance with MIQE guidelines gives researchers the tools to comprehensively document all aspects of their qPCR experiments, from sample preparation to data analysis. Thermo Fisher Scientific supports scientists and their adherence to the MIQE guidelines by offering products and resources that help facilitate compliance with these standards.

What are MIQE guidelines?

MIQE guidelines are a set of standards aimed at improving the quality and transparency of quantitative real-time PCR experiments. These guidelines cover all aspects of qPCR, including experimental design, sample quality, assay validation, and data analysis. By following MIQE, researchers can see to it that their experiments are well-documented and that their results can be independently verified by other scientists. This level of rigor is essential for advancing scientific knowledge and maintaining the integrity of research findings. Thermo Fisher Scientific supports scientists and their adherence to the MIQE guidelines by offering high-quality reagents, instruments, and software that facilitate compliance with these standards. Our comprehensive resources help researchers design accurate and efficient qPCR assays, supporting the credibility and reproducibility of scientific research. Thermo Fisher Scientific provides the assay ID along with an amplicon context sequence which is compliant with the MIQE guidelines 2.0 [1,2].

Comply with MIQE guidelines on assay sequence disclosure

In the case of predesigned Applied Biosystems TaqMan assays, >296K citations in articles in well-regarded, peer-reviewed publications [3,4,5], usually only reference the TaqMan Assay ID. Thus, publication of a unique identifier such as the Assay ID in lieu of sequences is typically sufficient and widely accepted. However, to fully comply with MIQE guidelines on assay sequence disclosure, the probe or amplicon context sequence in addition to the Assay ID will need to be provided.

  • The amplicon context sequence contains the full PCR amplicon.
  • The probe context sequence is a central sequence that contains the full probe sequence.
How to access your probe context sequence in the Assay Information File (AIF)*

Thermo Fisher Scientific provides a comprehensive Assay Information File (AIF) for each assay shipped that contains the required context sequence. Follow the steps below to access your AIF:

  1. Go to TaqMan File Downloads on thermofisher.com.
  2. Choose the radio button for “TaqMan Assays”.
  3. Enter the Sales Order # and Rack/Plate ID.
  4. A .zip folder will automatically download.
  5. Unzip the folder. The .txt and .html files contain the context sequence.
How to generate probe and amplicon context sequences for your TaqMan Assay*

The amplicon context sequence is easily found using the TaqMan Assay Search Tool using the following instructions. If needed, the probe context sequence can be generated using the same process.

It’s important to note that the primer/probe sequence within a TaqMan Assay ID will never change, ensuring the assay reference remains relevant over time.

  1. Enter the Assay ID of interest into the TaqMan Assay Search Tool.
  2. When you receive the search results, click on “Details”.
  3. Record any of the RefSeq Accession numbers listed.
  4. Also capture the Assay Location and Amplicon Length for that specific RefSeq Accession number.
  5. Extend the Assay Location:
    For a probe context sequence:
    Subtract 12 from and add 12 to the assay location (e.g., 1026 – 12 = 1014/1026 + 12 = 1038)

    For an amplicon context sequence:
    Subtract and add the amplicon length from and to the assay location
    (e.g., 1026 – 101 = 925/1026 + 101 = 1127)

Copy this URL to generate probe or amplicon context sequence:

https://www.ncbi.nlm.nih.gov/nuccore/
REFSEQ ID?report=fasta&log$=seqview&format
=text&from=XXX&to=XXX

  1. Enter the RefSeq and location values into the provided URL per the following examples:
    Probe context sequence:
    https://www.ncbi.nlm.nih.gov/nuccore/NM_001128128.2?report=fasta&log$=seqview&format=text&from=1014&to=1038 

    Amplicon context sequence:
    https://www.ncbi.nlm.nih.gov/nuccore/NM_001128128.2?report=fasta&log$=seqview&format=text&from=925&to=1127

  2. Copy/paste URL with the appropriate RefSeq and location values into an internet browser and hit enter. The sequence is generated.
    Probe context sequence:
    red box indicating where the probe context sequence is located
    Amplicon context sequence:
    red box indicating where the amplicon context sequence is located

*Instructions for generating a probe/amplicon context sequence do not apply to microbe or antibiotic resistance assays.

Information provided by TaqMan Assay search tool

The TaqMan Assay Search Tool serves up a host of helpful information, including the Entrez Gene ID, the gene symbol, a list of RefSeq IDs that are interrograted by that assay, and GenBank mRNA accession numbers. And while we don't share the specific oligo sequences, we provide the amplicon length, the probe location, and—if appropriate—the exon-exon boundary that the assay crosses. Finally, you can consult an assay map showing how all assays for a given gene line up with all known transcripts.

Assay annotation information requested under the qPCR target, qPCR oligonucleotide, and qPCR protocol sections of the guidelines are readily available on the Thermo Fisher Scientific website, and biologically relevant TaqMan Assay annotation information is included in the AIF.

References
  1. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Primer sequence disclosure: a clarification of the MIQE guidelines. Clin Chem. 2011 Jun;57(6):919–21. 
  2. Bustin SA, Ruijter JM, van den Hoff MJB, et al. MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines. Clin Chem. Published online April 24, 2025:hvaf043. 
  3. Mesko B, Poliskal S, Szegedi A, et al. Peripheral blood gene expression patterns discriminate among chronic inflammatory diseases and healthy controls and identify novel targets. BMC Med Genomics. 2010;3(1):15. 
  4. Sørlie T, Wang Y, Xiao C, et al. Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms. BMC Genomics. 2006;7(1):127. 
  5. Barr TL, Conley Y, Ding J, et al. Genomic biomarkers and cellular pathways of ischemic stroke by RNA gene expression profiling. Neurology. 2010;75(11):1009–1014. 
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