Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

B's PT Government Associate Laboratory Braga Guimarães Portugal

Berlin Institute of Health Germany

Brazilian Biosciences National Laboratory Campinas Brazil

CellCarta Zwijnaarde Belgium

Center for Cancer and Immunology Research Children's National Hospital Washington DC USA

City of Hope Comprehensive Cancer Center Duarte CA USA

Department of Biology Faculty of Medicine Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biomolecular Medicine Ghent University Belgium

Department of Comprehensive Cancer Care Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Medical and Surgical Sciences University of Bologna Italy

Department of Medical Biology Amsterdam UMC location AMC University of Amsterdam The Netherlands

Department of Medical Biology Faculty of Health Sciences UiT The Arctic University of Norway Tromsø Norway

Department of Molecular Diagnostics and Experimental Therapeutics Beckman Research Institute of City of Hope Monrovia CA USA

Department of Pharmaceutics University of Florida College of Pharmacy Gainesville FL USA

Department of Radiation Oncology State Key Laboratory of Oncology in South China Collaborative Innovation Center for Cancer Medicine Sun Yat sen University Cancer Center Guangzhou China

Department of Surgery Kyushu University Beppu Hospital Japan

Department of Translational Molecular Pathology The University of Texas MD Anderson Cancer Center Houston TX USA

Division of Animal Physiology and Immunology School of Life Sciences Weihenstephan Technical University of Munich Freising Germany

Division of Oncology Department of Internal Medicine Medical University of Graz Austria

Editorial Manager Molecular Oncology Cambridge UK

Genomics Core Facility European Molecular Biology Laboratory Heidelberg Germany

German Cancer Research Center Heidelberg Germany

Institute of Biotechnology CAS v v i Vestec Czech Republic

Institute of Pathology Berlin Institute of Health Charité Universitätsmedizin Berlin Corporate Member of Freie Universität Berlin Humboldt Universität zu Berlin Germany

IRCCS Azienda Ospedaliero Universitaria di Bologna Italy

Laboratory for Technologies of Advanced Therapies Department of Morphology Surgery and Experimental Medicine University of Ferrara Italy

Laboratory of Medical Genomics Centro Internacional de Pesquisa AC Camargo Cancer Center São Paulo Brazil

Laboratory of Neurosciences Institute of Psychiatry Faculdade de Medicina Universidade de São Paulo Brazil

Life and Health Sciences Research Institute School of Health Sciences University of Minho Braga Portugal

Medical Technology Research Centre Anglia Ruskin University Chelmsford Essex UK

Molecular Oncology Research Center Barretos Cancer Hospital Brazil

National Measurement Laboratory LGC Teddington UK

New South Wales Department of Primary Industries Elizabeth Macarthur Agricultural Institute Narellan Australia

OncoRNALab Cancer Research Institute Ghent Belgium

School of Biosciences and Medicine Faculty of Health and Medical Sciences University of Surrey Guildford UK

TATAA Biocenter AB Göteborg Sweden

The Center for Non codingRNAs University of Texas MD Anderson Cancer Center Houston TX USA

Translational Oncology University Hospital of Augsburg Stenglinstrasse Germany

Zentrum für Molekulare Biologie der Universität Heidelberg Germany

Zobrazit více v PubMed

Taylor SC, Nadeau K, Abbasi M, Lachance C, Nguyen M, Fenrich J. The ultimate qPCR experiment: producing publication quality, reproducible data the first time. Trends Biotechnol. 2019;37(7):761–74. 10.1016/j.tibtech.2018.12.002 PubMed DOI

Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real‐time RT‐PCR. Nat Protoc. 2006;1(3):1559–82. 10.1038/nprot.2006.236 PubMed DOI

Pfaffl MW. A new mathematical model for relative quantification in real‐time RT‐PCR. Nucleic Acids Res. 2001;29(9):e45. 10.1093/nar/29.9.e45 PubMed DOI PMC

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real‐time quantitative PCR and the 2(‐Delta Delta C(T)) method. Methods. 2001;25(4):402–8. 10.1006/meth.2001.1262 PubMed DOI

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real‐time quantitative RT‐PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034. 10.1186/gb-2002-3-7-research0034 PubMed DOI PMC

Bustin SA, Benes V, Garson J, Hellemans J, Huggett J, Kubista M, et al. The need for transparency and good practices in the qPCR literature. Nat Methods. 2013;10(11):1063–7. 10.1038/nmeth.2697 PubMed DOI

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real‐time PCR experiments. Clin Chem. 2009;55(4):611–22. 10.1373/clinchem.2008.112797 PubMed DOI

Gratz C, Bui MLU, Thaqi G, Kirchner B, Loewe RP, Pfaffl MW. Obtaining reliable RT‐qPCR results in molecular diagnostics‐MIQE goals and pitfalls for transcriptional biomarker discovery. Life (Basel). 2022;12(3):386. 10.3390/life12030386 PubMed DOI PMC

Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A practical approach to RT‐qPCR‐publishing data that conform to the MIQE guidelines. Methods. 2010;50(4):S1–5. 10.1016/j.ymeth.2010.01.005 PubMed DOI

Ruijter JM, Barnewall RJ, Marsh IB, Szentirmay AN, Quinn JC, van Houdt R, et al. Efficiency correction is required for accurate quantitative PCR analysis and reporting. Clin Chem. 2021;67(6):829–42. 10.1093/clinchem/hvab052 PubMed PMID: 33890632. PubMed DOI

Ruiz‐Villalba A, Ruijter JM, van den Hoff MJB. Use and misuse of C(q) in qPCR data analysis and reporting. Life (Basel). 2021;11(6):496. 10.3390/life11060496 PubMed DOI PMC

Errington TM, Mathur M, Soderberg CK, Denis A, Perfito N, Iorns E, et al. Investigating the replicability of preclinical cancer biology. Elife. 2021;10:e71601. 10.7554/eLife.71601 PubMed DOI PMC

Errington TM, Denis A, Allison AB, Araiza R, Aza‐Blanc P, Bower LR, et al. Experiments from unfinished registered reports in the reproducibility project: cancer biology. Elife. 2021;10:e73430. 10.7554/eLife.73430 PubMed DOI PMC

Mullard A. Half of top cancer studies fail high‐profile reproducibility effort. Nature. 2021;600(7889):368–9. 10.1038/d41586-021-03691-0 PubMed DOI

Baker M. 1,500 scientists lift the lid on reproducibility. Nature. 2016;533(7604):452–4. 10.1038/533452a PubMed DOI

Baker M. Reproducibility crisis: blame it on the antibodies. Nature. 2015;521(7552):274–6. 10.1038/521274a PubMed DOI

Ruijter JM, Ruiz‐Villalba A, van den Hoff MJB. Cq values do not reflect nucleic acid quantity in biological samples. Clin Chem. 2021;68(1):7–9. 10.1093/clinchem/hvab236 PubMed DOI

Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, et al. RDML: structured language and reporting guidelines for real‐time quantitative PCR data. Nucleic Acids Res. 2009;37(7):2065–9. 10.1093/nar/gkp056 PubMed DOI PMC