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Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond
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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 12586

Special Issue Editor

Prof. Dr. Stephen Bustin

E-Mail Website
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
Special Issues, Collections and Topics in MDPI journals

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

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

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.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • MIQE
  • qPCR
  • reverse transcription

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Published Papers (6 papers)

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Research

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32 pages, 2188 KB  
Article
Implementation and Validation of a Limiting Component Quantification Method for qPCR
by Andreas Untergasser, Quinn D. Gunst, Vladimir Benes and Maurice J. B. van den Hoff
Int. J. Mol. Sci. 2026, 27(6), 2717; https://doi.org/10.3390/ijms27062717 - 16 Mar 2026
Viewed by 582
Abstract
Quantitative polymerase chain reaction (qPCR) is a widespread method to quantify RNA or DNA. The results are reported as cycle of quantification (Cq), scaled to absolute numbers of copies or relative to reference genes. The reported Cq values of the same reaction vary [...] Read more.
Quantitative polymerase chain reaction (qPCR) is a widespread method to quantify RNA or DNA. The results are reported as cycle of quantification (Cq), scaled to absolute numbers of copies or relative to reference genes. The reported Cq values of the same reaction vary between different machines and cannot be compared between different laboratories. This study shows that the third derivative zero (TD0) method is machine independent and more reproducible than the classic Cq calculations. Together with the mean PCR efficiency it allows the calculation of the number of copies initially present (Ncopy), a parameter easy to interpret. A large dataset was created for the evaluation of this method including amplicons with different length, primer concentrations, reaction mixes, and fluorescence reporter systems. Furthermore, the calculated Ncopy values can be corrected at the same time using known concentrations of a standard and for the expression of reference genes and combining absolute and relative quantification. The algorithms were implemented in the open-source program RDML-Tools, which can perform all steps of a qPCR analysis using the raw fluorescence amplification data and is available on the internet. We conclude that qPCR analysis today should widen its focus and include the three essential parameters, TD0, mean PCR efficiency and Ncopy. Full article
(This article belongs to the Special Issue Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond)
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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
Cited by 4 | Viewed by 3395
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
(This article belongs to the Special Issue Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond)
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Review

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20 pages, 3652 KB  
Review
A Memoir of Inventing Real-Time PCR and Developing the ABI 7700
by Russell Higuchi and Lincoln McBride
Int. J. Mol. Sci. 2026, 27(6), 2612; https://doi.org/10.3390/ijms27062612 - 12 Mar 2026
Viewed by 1217
Abstract
Real-time PCR (qPCR) is today’s definitive quantitative technology in molecular biology and diagnostics. Until 30 years ago, PCR product analyses were generally performed after amplification using gel-based methods. Quantification typically relied on visual inspection or densitometry of end-point products and was therefore relatively [...] Read more.
Real-time PCR (qPCR) is today’s definitive quantitative technology in molecular biology and diagnostics. Until 30 years ago, PCR product analyses were generally performed after amplification using gel-based methods. Quantification typically relied on visual inspection or densitometry of end-point products and was therefore relatively unreliable and poorly suited to high-throughput automation. To celebrate real-time PCR’s 30-year anniversary of commercial availability, Professor Stephen Bustin, Guest Editor for the special edition, “Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond,” asked Russell Higuchi to give a historical account on how his idea of real-time PCR was conceived and brought to fruition. Dr. Higuchi then asked his collaborator, Lincoln McBride, who drove the development of the ABI 7700—the high-throughput real-time PCR instrument that gave researchers access to this technology—to co-author this dual memoir. This story is told from the perspectives of the two scientists most directly responsible for making real-time PCR practical and widely accessible. Taking turns, Russell Higuchi describes the conceptual and experimental steps at Cetus and then Roche that led from homogeneous PCR detection to continuous fluorescence monitoring, whilst Lincoln McBride details ABI’s parallel efforts to commercialize Russ’s invention. Together, they trace how experimental insight, engineering constraints, product development, and commercial decision-making shaped the Applied Biosystems 7700 Sequence Detection System and established real-time PCR as a practical and reliable quantitative technology. Their team’s efforts persevered through technological uncertainty and within a complex corporate collaboration. They share key historical documents in their original form. Their accounts show how the 7700 system emerged as the convergence of chemistry, optics, software, and product development. The eventual global reliance on real-time PCR during the COVID-19 pandemic demonstrated, at unprecedented scale, the profound and enduring impact of these early technical and organizational choices. Full article
(This article belongs to the Special Issue Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond)
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23 pages, 1535 KB  
Review
Quantification Revisited: What qPCR Efficiency Models Reveal About Data Analysis Integrity
by Stephen A. Bustin, Maurice J. B. van den Hoff, Michael W. Pfaffl, Mikael Kubista and Jan M. Ruijter
Int. J. Mol. Sci. 2026, 27(5), 2337; https://doi.org/10.3390/ijms27052337 - 2 Mar 2026
Viewed by 907
Abstract
Amplification efficiency is one of the key parameters in quantitative real-time PCR, as it directly influences the accuracy of both absolute and relative quantification. Amplification efficiency, the fold increase per cycle, is affected by oligonucleotide design, reaction chemistry, sample and template properties, and [...] Read more.
Amplification efficiency is one of the key parameters in quantitative real-time PCR, as it directly influences the accuracy of both absolute and relative quantification. Amplification efficiency, the fold increase per cycle, is affected by oligonucleotide design, reaction chemistry, sample and template properties, and instrument performance. Consequently, it differs between samples, assays and experimental runs. Although methods for estimating the amplification efficiency have been available for more than two decades, most published qPCR studies continue to assume equal and ideal efficiency across assays. This simplifying assumption introduces efficiency- and expression-dependent error into relative expression and fold-change analyses, contributing to the poor reproducibility observed in many PCR-based studies. This review examines the role of the amplification efficiency in qPCR quantification; the consequences of ignoring, assuming or misapplying efficiency; and the practical implications for data interpretation. The review also considers the development of efficiency estimation models and their implications for contemporary analytical approaches, including emerging data-driven methods for amplification curve analyses. Full article
(This article belongs to the Special Issue Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond)
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21 pages, 1086 KB  
Review
Analysis of qPCR Data: From PCR Efficiency to Absolute Target Quantity
by Jan M. Ruijter and Maurice J. B. van den Hoff
Int. J. Mol. Sci. 2025, 26(24), 11885; https://doi.org/10.3390/ijms262411885 - 9 Dec 2025
Cited by 2 | Viewed by 3428
Abstract
Quantitative Polymerase Chain Reaction (qPCR) is a very sensitive method to determine small amounts of DNA or RNA in experimental, environmental, veterinary, forensic and clinical samples. Despite efforts from the qPCR community to address qPCR variability by recommending standardization of reporting of all [...] Read more.
Quantitative Polymerase Chain Reaction (qPCR) is a very sensitive method to determine small amounts of DNA or RNA in experimental, environmental, veterinary, forensic and clinical samples. Despite efforts from the qPCR community to address qPCR variability by recommending standardization of reporting of all steps of a qPCR experiment, most reported qPCR results are still grossly biased. The first part of this paper describes two decades of efforts to remedy this situation by promoting so-called efficiency-corrected qPCR data analysis. Although such analysis leads to less variable qPCR results, the outcome, fluorescence at cycle zero, is difficult to grasp. In the second part, we outline how qPCR analyses can result in Ncopy, the number of copies of the target at the start of the reaction. A newly developed theoretical approach determines Ncopy using the characteristics of the amplification curve and the known concentrations of all reaction components. By including these reaction-mix characteristics in the analysis, this Ncopy is assay-, machine- and laboratory-independent and thus allows direct worldwide comparisons. Moreover, Ncopy provides a very intuitive and easy-to-interpret absolute quantitative result. Full article
(This article belongs to the Special Issue Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond)
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Other

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10 pages, 1519 KB  
Commentary
How Laboratory Guidelines Promote the Validity of Circulating Extracellular Vesicle-Associated Nucleic Acid Biomarker Signatures in Liquid Biopsy
by Michael W. Pfaffl
Int. J. Mol. Sci. 2025, 26(24), 12115; https://doi.org/10.3390/ijms262412115 - 16 Dec 2025
Cited by 1 | Viewed by 782
Abstract
Circulating nucleic acids, particularly those associated with extracellular vesicles (EVs), represent a promising class of molecular biomarkers in liquid biopsy for ‘non-invasive’ disease diagnostics, for better prognosis, and for therapeutic monitoring. However, the translation of this new circulating biomarker source into clinical practice [...] Read more.
Circulating nucleic acids, particularly those associated with extracellular vesicles (EVs), represent a promising class of molecular biomarkers in liquid biopsy for ‘non-invasive’ disease diagnostics, for better prognosis, and for therapeutic monitoring. However, the translation of this new circulating biomarker source into clinical practice is mostly hindered by methodological variability and a lack of standardization across the analytical workflow. This article highlights the implementation of international academic guidelines, such as Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) and Minimal Information for Studies of Extracellular Vesicles (MISEV), in the entire analytical procedure in promoting the integrity, reproducibility, and validity of EV-associated nucleic acid markers in molecular diagnostics. By standardizing the liquid biopsy workflow from tissue sampling up to data analysis and statistics, these established guidelines lay the necessary scientific basis for a robust, reproducible, reliable, and valid RNA and DNA biomarker discovery in EVs. The ultimate goal is the successful implementation of the developed biomarker signature into the clinical diagnostic routine, but this requires further rounds of rigorous validation. The regularly updated guidelines should not be seen as optional recommendations, but more like an essential pillars of scientific rigor and standardization in order to achieve better and biological meaningful biomarker results in liquid biopsy. Full article
(This article belongs to the Special Issue Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond)
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