Papers in Press. Published February 26, 2009 as doi:10.1373/clinchem.2008.112797 The latest version is at http://www.clinchem.org/cgi/doi/10.1373/clinchem.2008.112797 Clinical Chemistry 55:4 000–000 (2009) Reviews The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments Stephen A. Bustin,1* Vladimir Benes,2 Jeremy A. Garson,3,4 Jan Hellemans,5 Jim Huggett,6 Mikael Kubista,7,8 Reinhold Mueller,9 Tania Nolan,10 Michael W. Pfaffl,11 Gregory L. Shipley,12 Jo Vandesompele,5 and Carl T. Wittwer13,14 BACKGROUND: Currently, a lack of consensus exists on SUMMARY: Following these guidelines will encourage how best to perform and interpret quantitative real- better experimental practice, allowing more reliable time PCR (qPCR) experiments. The problem is exac- and unequivocal interpretation of qPCR results. erbated by a lack of sufficient experimental detail in © 2009 American Association for Clinical Chemistry many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. The fluorescence-based quantitative real-time PCR (qPCR)15 (1–3), with its capacity to detect and mea- CONTENT: The Minimum Information for Publication sure minute amounts of nucleic acids in a wide range of of Quantitative Real-Time PCR Experiments (MIQE) samples from numerous sources, is the enabling tech- guidelines target the reliability of results to help ensure nology par excellence of molecular diagnostics, life sci- the integrity of the scientific literature, promote con- ences, agriculture, and medicine (4, 5). Its conceptual sistency between laboratories, and increase experimen- and practical simplicity, together with its combination tal transparency. MIQE is a set of guidelines that de- of speed, sensitivity, and specificity in a homogeneous scribe the minimum information necessary for assay, have made it the touchstone for nucleic acid evaluating qPCR experiments. Included is a checklist to quantification. In addition to its use as a research tool, accompany the initial submission of a manuscript to many diagnostic applications have been developed, in- the publisher. By providing all relevant experimental cluding microbial quantification, gene dosage determi- conditions and assay characteristics, reviewers can as- nation, identification of transgenes in genetically mod- sess the validity of the protocols used. Full disclosure of ified foods, risk assessment of cancer recurrence, and all reagents, sequences, and analysis methods is neces- applications for forensic use (6–11). sary to enable other investigators to reproduce results. This popularity is reflected in the prodigious MIQE details should be published either in abbreviated number of publications reporting qPCR data, which form or as an online supplement. invariably use diverse reagents, protocols, analysis methods, and reporting formats. This remarkable lack of consensus on how best to perform qPCR ex- periments has the adverse consequence of perpetu- ating a string of serious shortcomings that encumber 1 Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and its status as an independent yardstick (12).Techni- 2 the London School of Medicine and Dentistry, London, UK; Genomics Core cal deficiencies that affect assay performance include Facility, EMBL Heidelberg, Heidelberg, Germany; 3 Centre for Virology, Depart- ment of Infection, University College London, London, UK; 4 Department of the following: (a)inadequatesamplestorage,prep- Virology, UCL Hospitals NHS Foundation Trust, London, UK; 5 Center for Medical aration, and nucleic acid quality, yielding highly Genetics, Ghent University Hospital, Ghent, Belgium; 6 Centre for Infectious Diseases, University College London, London, UK; 7 TATAA Biocenter, Go¨ teborg, variable results; (b)poorchoiceofreverse-transcription Sweden; 8 Institute of Biotechnology AS CR, Prague, Czech Republic; 9 Seque- primers and primers and probes for the PCR, leading to nom, San Diego, California, USA; 10 Sigma–Aldrich, Haverhill, UK; 11 Physiology inefficient and less-than-robust assay performance; Weihenstephan, Technical University Munich, Freising, Germany; 12 Quantita- tive Genomics Core Laboratory, Department of Integrative Biology and Phar- macology, University of Texas Health Science Center, Houston, Texas, USA; 13 Department of Pathology, University of Utah, Salt Lake City, Utah, USA; 14 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA. 15 Nonstandard abbreviations: qPCR, quantitative real-time PCR; MIQE, Minimum * Address correspondence to this author at: 3rd Floor Alexandra Wing, The Royal Information for Publication of Quantitative Real-Time PCR Experiments; RT- London Hospital, London E1 1BB, UK. Fax ϩ44-(0)20-7377 7283; e-mail qPCR, reverse transcription–qPCR; FRET, fluorescence resonance energy trans- [email protected]. fer; Cq, quantification cycle, previously known as the threshold cycle (Ct), Received October 20, 2008; accepted January 27, 2009. crossing point (Cp), or take-off point (TOP); RDML, Real-Time PCR Data Markup Previously published online at DOI: 10.1373/clinchem.2008.112797 Language; LOD, limit of detection; NTC, no-template control. 1 Copyright (C) 2009 by The American Association for Clinical Chemistry Reviews and (c) inappropriate data and statistical analyses, gen- sion and is inconsistent with its use for conven- erating results that can be highly misleading. Conse- tional (legacy) reverse transcription–PCR. quently, there is the real danger of the scientific litera- 1.2 Genes used for normalization should be referred to ture being corrupted with a multitude of publications as reference genes, not as housekeeping genes. reporting statistically insignificant and conflicting re- 1.3 TaqMan probes should be referred to as hydrolysis sults ( 13). The publication (14) and retraction (15) of probes. a Science “Breakthrough of the Year 2005” report pro- 1.4 The term FRET probe (fluorescence resonance en- vides a disquieting warning. The problem is exacer- ergy transfer probe) refers to a generic mechanism bated by the lack of information that characterizes in which emission/quenching relies on the interac- most reports of studies that have used this technol- tion between the electron-excitation states of 2 flu- ogy, with many publications not providing sufficient orescent dye molecules. LightCycler-type probes experimental detail to permit the reader to critically should be referred to as dual hybridization probes. evaluate the quality of the results presented or to 1.5 The Oxford English Dictionary lists only quantifica- repeat the experiments. Specifically, information tion, not quantitation; therefore, the former is the about sample acquisition and handling, RNA quality proper word. and integrity, reverse-transcription details, PCR ef- 1.6 The nomenclature describing the fractional PCR ficiencies, and analysis parameters are frequently cycle used for quantification is inconsistent, with omitted, whereas sample normalization is habitually threshold cycle (Ct), crossing point (Cp), and take-off carried out against single reference genes without point (TOP) currently used in the literature. These adequate justification. terms all refer to the same value from the real-time The aim of this document is to provide authors, instrument and were coined by competing manu- reviewers, and editors specifications for the minimum facturers of real-time instruments for reasons of information, set out in Table 1, that must be reported product differentiation, not scientific accuracy or for a qPCR experiment to ensure its relevance, accu- clarity. We propose the use of quantification cycle racy, correct interpretation, and repeatability. MIQE (Cq), according to the RDML (Real-Time PCR (Minimum Information for Publication of Quantita- Data Markup Language) data standard (http:// tive Real-Time PCR Experiments, pronounced mykee) www.rdml.org) (27). is modeled on similar guidelines drawn up for DNA microarray analysis (16), proteomics experiments 2. Conceptual Considerations (17), genome sequence specification (18), and those under discussion for RNA interference work (19, 20) To explain and justify the guidelines, we find it useful and metabolomics (21), all of which are initiatives co- to review a number of key issues surrounding qPCR ordinated under the umbrella of MIBBI (Minimum experiments: Information for Biological and Biomedical Investiga- tions, http://www.mibbi.org) (22). Compulsory inclu- 2.1 Analytical sensitivity refers to the minimum num- sion of a common reporting language to allow data ber of copies in a sample that can be measured sharing is not proposed, although it is envisaged that a accurately with an assay, whereas clinical sensitivity future update of these guidelines could include such a is the percentage of individuals with a given disor- recommendation. Rather, these guidelines target the der whom the assay identifies as positive for that reliability of results to help ensure the integrity of the condition. Typically, sensitivity is expressed as the scientific literature, promote consistency between lab- limit of detection (LOD), which is the concentra- oratories, and increase experimental transparency. tion that can be detected with reasonable certainty They should be read in conjunction with recent publi- (95% probability is commonly used) with a given cations that deal in depth with the issue of qPCR stan- analytical procedure. The most sensitive