The Need for Transparency and Good Practices in the QPCR Literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Citing Articles

Development, Optimization, and Validation of a Quantitative PCR Assay for Detection in Tick, Wildlife, and Human Samples.

Lewis J, Lloyd V, Robichaud G Pathogens. 2025; 13(12.

PMID: 39770294 PMC: 11679815. DOI: 10.3390/pathogens13121034.

Selection and validation of reference genes for qRT-PCR normalization in dayflower (Commelina communis) based on the transcriptome profiling.

Yang Y, Cao G, Tang L BMC Plant Biol. 2024; 24(1):1131.

PMID: 39592924 PMC: 11600965. DOI: 10.1186/s12870-024-05853-4.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

Sadikan M, Abdul Nasir N, Ibahim M, Iezhitsa I, Agarwal R Int J Ophthalmol. 2024; 17(5):794-805.

PMID: 38766348 PMC: 11074185. DOI: 10.18240/ijo.2024.05.02.

Low-Density TaqMan® Array Cards for the Detection of Pathogens.

Heaney J, Rolfe K, Gleadall N, Greatorex J, Curran M Methods Microbiol. 2024; 42:199-218.

PMID: 38620215 PMC: 7172410. DOI: 10.1016/bs.mim.2015.06.002.

Comprehensive Assessment of Reference Gene Expression within the Whitefly Using RT-qPCR.

Kong W, Lv X, Ran X, Mukangango M, Eric Derrick B, Qiu B Genes (Basel). 2024; 15(3).

PMID: 38540377 PMC: 10970672. DOI: 10.3390/genes15030318.

References

Bustin S . Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000; 25(2):169-93. DOI: 10.1677/jme.0.0250169. View

Derveaux S, Vandesompele J, Hellemans J . How to do successful gene expression analysis using real-time PCR. Methods. 2009; 50(4):227-30. DOI: 10.1016/j.ymeth.2009.11.001. View

Garson J, Huggett J, Bustin S, Pfaffl M, Benes V, Vandesompele J . Unreliable real-time PCR analysis of human endogenous retrovirus-W (HERV-W) RNA expression and DNA copy number in multiple sclerosis. AIDS Res Hum Retroviruses. 2009; 25(3):377-8. DOI: 10.1089/aid.2008.0270. View

Bustin S, Penning L . Improving the analysis of quantitative PCR data in veterinary research. Vet J. 2011; 191(3):279-81. DOI: 10.1016/j.tvjl.2011.06.044. View

Bustin S . Why the need for qPCR publication guidelines?--The case for MIQE. Methods. 2009; 50(4):217-26. DOI: 10.1016/j.ymeth.2009.12.006. View

Bustin S . Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences. Expert Rev Mol Diagn. 2005; 5(4):493-8. DOI: 10.1586/14737159.5.4.493. View

Fleige S, Pfaffl M . RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006; 27(2-3):126-39. DOI: 10.1016/j.mam.2005.12.003. View

Kubista M, Andrade J, Bengtsson M, Forootan A, Jonak J, Lind K . The real-time polymerase chain reaction. Mol Aspects Med. 2006; 27(2-3):95-125. DOI: 10.1016/j.mam.2005.12.007. View

Bustin S, Beaulieu J, Huggett J, Jaggi R, Kibenge F, Olsvik P . MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. 2010; 11:74. PMC: 2955025. DOI: 10.1186/1471-2199-11-74. View

10.

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

11.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

12.

Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M . Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem. 2002; 309(2):293-300. DOI: 10.1016/s0003-2697(02)00311-1. View

13.

Horrobin D . Modern biomedical research: an internally self-consistent universe with little contact with medical reality?. Nat Rev Drug Discov. 2003; 2(2):151-4. DOI: 10.1038/nrd1012. View

14.

Scholl C, Frohling S, Dunn I, Schinzel A, Barbie D, Kim S . Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell. 2009; 137(5):821-34. DOI: 10.1016/j.cell.2009.03.017. View

15.

Ioannidis J, Khoury M . Improving validation practices in "omics" research. Science. 2011; 334(6060):1230-2. PMC: 5624327. DOI: 10.1126/science.1211811. View

16.

Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S . Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res. 2011; 39(9):e63. PMC: 3089491. DOI: 10.1093/nar/gkr065. View

17.

Huggett J, Dheda K, Bustin S, Zumla A . Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005; 6(4):279-84. DOI: 10.1038/sj.gene.6364190. View

18.

Pfaffl M . A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29(9):e45. PMC: 55695. DOI: 10.1093/nar/29.9.e45. View

19.

Babij C, Zhang Y, Kurzeja R, Munzli A, Shehabeldin A, Fernando M . STK33 kinase activity is nonessential in KRAS-dependent cancer cells. Cancer Res. 2011; 71(17):5818-26. DOI: 10.1158/0008-5472.CAN-11-0778. View

20.

Bustin S . Transparency of reporting in molecular diagnostics. Int J Mol Sci. 2013; 14(8):15878-84. PMC: 3759891. DOI: 10.3390/ijms140815878. View