In April 2025, the MIQE guidelines for quantitative real-time PCR (qPCR) were updated for the first time since their initial release in 2009. This latest publication addresses the many advances in qPCR methods since then.
The first MIQE (Minimum Information for publication of Quantitative real-time PCR Experiments) guidelines by Bustin et al. sought to improve qPCR experiments and allow more reliable interpretation of results.
The international team of researchers identified that there was a lack of detail about the experiments carried out in many publications. This prevented other researchers from evaluating or replicating the findings.
To address this, Bustin et al. wrote the MIQE (pronounced my-kee) guidelines for the minimum details required for analysing qPCR experiments. This aimed to create a more consistent format for reporting across the wide range of qPCR protocols. The paper included an 85-point checklist, which has been adopted as part of the submission process by some journals.
The MIQE guidelines established standardised nomenclature, optimisation, validation and analysis for qPCR. This formed the basis for the ISO20395:2019 requirements for evaluating the performance of quantification methods for nucleic acid target sequences.
Since 2009, however, qPCR has changed dramatically. There are a vast array of oligo modifications available, along with improvements in reagents, throughput, precision and methods.
Updates in MIQE 2.0 for qPCR
MIQE 2.0 expands on the previous iteration by addressing the key factors that affect the quality of qPCR experiments. This represents a subtle shift in the approach of MIQE, from focusing primarily on reporting criteria to placing greater emphasis on good practice for assay design.
The reporting checklist has been streamlined, making it easier for researchers to adhere to it. However, there are some key additions, including:
- Publishing confidence intervals for the limit of detection, limits of quantification, PCR efficiency and other critical figures
- Making raw data available for verification and re-analysis
- Converting Cq values to efficiency-corrected target quantities
MIQE 2.0 provides more detailed guidance on sample handling, assay optimisation and validation protocols. Many of its recommendations on assay design and analysis strengthen previous advice across several aspects, including:
- Using multiple negative controls and exogenous spike-ins
- Ensuring proper sample storage and maintaining its integrity
- Adopting more rigorous approaches to data manipulation, including normalisation, baseline correction and outlier detection
Relevance of MIQE 2.0 for qPCR in clinical diagnostics
MIQE 2.0 specifically addresses several key considerations for ensuring reliable results in qPCR assays for clinical diagnostics.
The paper provides an updated perspective on sample preparation following the COVID-19 pandemic. The demand for rapid diagnostics prompted significant growth in direct RT-qPCR using crude samples. This was made possible by enzyme formulations that are resistant to inhibitors. However, delivering accurate results with this streamlined method requires further optimisation and validation to manage the variability in RNA quantity and quality.
In addition, the MIQE 2.0 authors highlight the common practice of reporting only Cq values in diagnostic tests. However, Cq can depend on several analysis steps, including baseline subtraction, choice of quantification threshold and smoothing algorithm usage.
Furthermore, the updated guidelines advise against conducting statistical tests of Cq values, as they are neither efficiency-corrected nor normalised. To avoid this, Cq values should be converted to target quantities by comparing the results with those from a known standard.
MIQE 2.0 also highlights the growing use of multiplex assays in clinical testing. By adding reference genes or an external template as in-sample controls, this approach can confirm the robustness of the sample preparation workflow and validate a negative test result. For more information about designing multiplex qPCR assays, download our ebook.
Our latest guide for optimisation and validation of custom oligonucleotides is also now available.
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To learn more about optimising your multiplex assays, download our latest ebook. 
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References
- Bustin SA et al. (2025) MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines. Clinical Chemistry hvaf043 doi: 1093/clinchem/hvaf043
- Bustin SA et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55:611–22 doi: 1373/clinchem.2008.112797
