Article 
Introducing MIQE and dMIQE Guidelines
In 2009, Stephen Bustin and his colleagues published the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in the Clinical Chemistry journal.1 The MIQE guidelines outline the minimum information that must be reported to ensure a qPCR experiment's relevance, accuracy, correct interpretation, and repeatability. In addition to a detailed checklist of experimental parameters to monitor and report results, the MIQE guidelines also offer recommendations for defining and using terminology. It strives to improve both how qPCR experiments are conducted and communicated.
Similarly, Jim Huggett and colleagues developed parallel guidelines for digital PCR (dPCR) experiments in 2013.2 These guidelines, called Minimum Information for Publication of Quantitative Digital PCR Experiments (dMIQE), were updated in 2020 by the same group to reflect advances in instrumentation and the broadening of recognized applications.3 Like the MIQE guidelines, the original and updated dMIQE publications also include a detailed checklist and standardized terminology recommendations for dPCR experiments and publications.
MIQE and dMIQE guidelines cover a range of topics, and they link everything together with one main underlying theme: thoroughness.
Reporting Sample Details
Concerning samples, researchers should provide information on the source, processing, storage, and composition, as well as nucleic acid quantity and quality assessment results. Minimum quality thresholds for qPCR and ddPCR should be disclosed. Moreover, while the reverse transcription (RT) step of RT-qPCR and RT-ddPCR is often overlooked as a means to convert RNA to cDNA, it can introduce substantial variation. MIQE and dMIQE guidelines advocate that this step be fully documented3 and carried out in duplicate or triplicate.1
Assay Design
For the actual qPCR or ddPCR assay, researchers should provide sufficient information on both experimental parameters and reagents to allow for replication. This includes database accession numbers for each target and reference gene, specificity assessments for each primer or probe, and sequences and concentrations of each oligonucleotide used (including the identities, positions, and linkages of any dyes and/or modified bases). The MIQE and dMIQE guidelines also state that the PCR reaction mixture should be detailed, including the concentrations and identity of the polymerase used, the amount of template in each reaction, and the compositions, volumes, and concentrations for other reaction elements such as Mg(2+), salts, and any additives. Finally, investigators must identify the instrument that performed the cycling, as well as the properties and manufacturers of the consumables that they used, including tubes and plates.1, 3
Data Analysis
In terms of data rigor, analysis, and presentation, the MIQE and dMIQE guidelines provide detailed guidance on calibration, normalization, and controls. In particular, the guidelines highlight how researchers must compensate for potential amplification efficiency differences between different primer sets and genes of interest.1, 3 Here, software programs and suites offer researchers a way to perform all necessary calibrations and standardizations to limit variability and promote consistency. Modern qPCR and ddPCR software packages can map calibration and standardization results onto experimental data, allowing automatic adjustments to account for reaction efficiency and comparisons to multiple reference markers. They can even combine experimental data from multiple plates and collate them for larger gene studies.4
Publishing Valid and Translatable Results
Overall, MIQE and dMIQE guidelines provide the resources scientists need to design, execute, and report nucleic acid quantification experiments that are of the highest scientific quality and are maximally reproducible. As the implementation of these guidelines continues to become more widespread, studies that include qPCR or ddPCR workflows will produce results with more trusted validity, which in turn will provide translational biological insights that inform in vitro diagnostic testing, therapeutic development, and quality control, and much more.
With years of experience implementing these PCR best practices, Bio-Rad experts are ready to help you do the same for your unique research.
References
- S.A. Bustin et al., “The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments,” Clin Chem, 55(4):611-22, 2009.
- J. F. Huggett et al., “The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments” Clin Chem, 59(6):892-902, 2013.
- J. F. Huggett et al., “The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020,” Clin Chem, 66(8):1012-1029, 2020.
- S. Taylor et al., “A practical approach to RT-qPCR—publishing data that conform to MIQE guidelines,” Bulletin 5859 Ver. E, Bio-Rad Laboratories, Inc., Hercules, CA, USA. https://www.bio-rad.com/sites/default/files/2021-09/Bulletin_5859.pdf
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