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Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: 20 May 2026 | Viewed by 1960

Special Issue Editor


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Guest Editor
Medical Technology Research Institute, Anglia Ruskin University, Chelmsford CM1 1SQ, UK
Interests: qPCR; RT-qPCR; colorectal cancer; molecular staging; clostridium difficile; MRSA; aspergillus
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Special Issue Information

Dear Colleagues,

Quantitative PCR (qPCR) remains one of the most powerful and widely used tools in molecular science, impacting an extraordinary range of fields, including basic and translational research, clinical diagnostics, forensics, agriculture, environmental science, biotechnology, and ancestry testing. Its reach is matched by its adaptability—but also by a persistent vulnerability to methodological inconsistency and a lack of transparency, which continue to undermine confidence in published results.
The Minimum Information for Publication of Quantitative PCR Experiments (MIQE) guidelines were first introduced in 2009 in response to the widespread misuse of qPCR and, in part, to high-profile methodological failures such as the RT-qPCR-based claims surrounding the MMR vaccine and autism. MIQE established essential principles for assay design, validation, and reporting, promoting transparency, reproducibility, and interpretability. In light of technological advances and expanding diagnostic applications, these guidelines have now been comprehensively revised as MIQE 2.0, recently published in Clinical Chemistry. The updated framework reflects current capabilities while reaffirming that methodological rigour remains a prerequisite for credible molecular data.

This Special Issue of IJMS invites original research, reviews, technical reports, and commentaries that critically examine the role of MIQE in improving qPCR-based workflows and ensuring the robustness of derived conclusions. Submissions may address the following:

  • How MIQE has improved reproducibility, accuracy, and inter-laboratory consistency;
  • Empirical or theoretical insights into amplification efficiency, error propagation, reference gene normalisation, and assay validation;
  • The influence of MIQE across related technologies (including digital PCR) and in specialised applications such as single-cell analysis, low-input workflows, or environmental testing;
  • Constructive critiques of MIQE’s implementation, limitations, or scope, including reflections on barriers to adoption.
This IJMS Special Issue offers a timely platform to reaffirm the importance of methodological discipline in molecular research. At a time when trust in scientific data is strained—and when too many qPCR results remain irreproducible, opaque, or biologically implausible—MIQE 2.0 reasserts the need for transparency, validation, and critical self-scrutiny. We seek contributions that engage with these challenges constructively and help shape a renewed culture of rigour and reproducibility in molecular quantification.

Prof. Dr. Stephen Bustin
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

  • MIQE
  • qPCR
  • reverse transcription

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Published Papers (1 paper)

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Research

26 pages, 3160 KB  
Article
When Two-Fold Is Not Enough: Quantifying Uncertainty in Low-Copy qPCR
by Stephen A. Bustin, Sara Kirvell, Tania Nolan, Reinhold Mueller and Gregory L. Shipley
Int. J. Mol. Sci. 2025, 26(16), 7796; https://doi.org/10.3390/ijms26167796 - 12 Aug 2025
Viewed by 1176
Abstract
Accurate interpretation of qPCR data continues to present significant challenges, particularly at low target concentrations where technical variability, stochastic amplification, and efficiency fluctuations confound quantification. The widespread assumption that qPCR outputs are intrinsically reliable, coupled with inconsistent adherence to best-practice guidelines, has exacerbated [...] Read more.
Accurate interpretation of qPCR data continues to present significant challenges, particularly at low target concentrations where technical variability, stochastic amplification, and efficiency fluctuations confound quantification. The widespread assumption that qPCR outputs are intrinsically reliable, coupled with inconsistent adherence to best-practice guidelines, has exacerbated issues of reproducibility and contributed to misleading conclusions. This may distort pathogen load quantification in diagnostic settings, whilst in gene expression studies, it can lead to overinterpretation of small fold changes. This study presents a systematic, cross-platform evaluation of qPCR performance across a wide dynamic range using defined reaction mixes and technical replicates. We show that calculated copy numbers can closely match expected values over more than three orders of magnitude, but that variability increases markedly at low input concentrations, often exceeding the magnitude of biologically meaningful differences. We conclude that establishing and reporting confidence intervals from the data itself is essential for transparency and for distinguishing reliable quantification from technical noise. Full article
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