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TaqMan Assays comply with MIQE guidelines

MIQE, which stands for Minimum Information for Publication of Quantitative Real-Time PCR Experiments, is a set of guidelines for real-time PCR (qPCR) experimental designs and data reporting practices, as well as standards for sharing experimental information with colleagues. The standards are designed to ensure that published real-time PCR research is meaningful, accurate, and provides researchers with the information necessary to faithfully reproduce results.

Thermo Fisher Scientific supports MIQE guidelines and provides all information necessary to ensure MIQE compliance when publishing the results of your real-time PCR experiments.

There has been some confusion about this since, originally, MIQE guidelines stipulated that researchers publish the primer/probe sequences for the assays used. However, those sequences were subsequently recognized as being commercially sensitive, so there was a later amendment by the authors that stated that “EITHER primer sequences OR amplicon context sequence” (1) could be referenced.

Meeting MIQE guidelines

Assay design

In the case of predesigned Applied Biosystems TaqMan Assays, >290K citations in articles in well-regarded, peer-reviewed publications such as Nature, Science and Nature Methods, frequently 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, you’ll need to provide either a probe or amplicon context sequence in addition to the Assay ID.

  • 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)*

We provide 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 “View 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:

    Amplicon context sequence:

 

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

Information provided by TaqMan Assay search tool

COL32524-TaqMan-showcase

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 in the Thermo Fisher Scientific reagent protocol or from the Thermo Fisher Scientific website. Biologically relevant TaqMan Assay annotation information is included in the AIF.

Video: Do TaqMan Assays comply with MIQE Guidelines?

Learn how TaqMan Assays comply with the Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE) set of recommendations by watching our Ask TaqMan video.

Reference

  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.

For Research Use Only. Not for use in diagnostic procedures.