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Real-Time Reverse Transcription Quantitative PCR (RT-qPCR) Methodological Standards and Reporting Practices
Stephen A Bustin1, Carl T Wittwer2
1Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University, Chelmsford, United Kingdom.
Clinical Chemistry
|January 28, 2026
Summary
Methodological reporting in real-time reverse transcription quantitative PCR (RT-qPCR) assays remains poor, with critical parameters like RNA integrity and PCR efficiency declining significantly. Adherence to Minimum Information for Publication of Quantitative PCR Experiments (MIQE) guidelines is insufficient, impacting assay reproducibility.
Area of Science:
- Life Sciences
- Molecular Biology
- Biotechnology
Background:
- Concerns persist regarding the methodological quality and reporting transparency of real-time reverse transcription quantitative PCR (RT-qPCR).
- The COVID-19 pandemic highlighted these issues in diagnostic testing.
- The Minimum Information for Publication of Quantitative PCR Experiments (MIQE) guidelines, updated in 2025, have had a modest impact on standardizing assay reporting.
Purpose of the Study:
- To assess trends in RT-qPCR methodological reporting between 2007 and 2025.
- To evaluate the impact of MIQE guidelines on reporting quality.
- To compare reporting practices across different time points, regions, and MIQE citation status.
Main Methods:
- PubMed Central searches and manual evaluation of 355 full-text articles from 2019 and 2024.
- Analysis of parameters including RNA integrity, oligonucleotide disclosure, reference gene validation, and PCR efficiency.
- Evaluation of targeted cohorts focusing on reference genes and PCR efficiency.
Main Results:
- Reporting of core RT-qPCR parameters remained low or declined between 2019 and 2024.
- RNA integrity reporting decreased from 22% to 11%, reference gene validation from 13% to 5%, and PCR efficiency reporting from 13% to 1%.
- MIQE-citing papers in 2024 showed better adherence but still omitted essential details; Asia dominates RT-qPCR output, while Europe leads in MIQE citations.
Conclusions:
- Incomplete experimental design and reporting in RT-qPCR assays continue to compromise reproducibility and robustness.
- Despite updated MIQE guidelines, adherence to essential reporting standards remains inadequate.
- Further efforts are needed to improve methodological quality and transparency in RT-qPCR publications.
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