Real-Time Reverse Transcription Quantitative PCR (RT-qPCR) Methodological Standards and Reporting Practices

Real-time reverse transcription quantitative PCR (RT-qPCR) is utilized in many areas of the life sciences, diagnostics, and forensics, yet concerns about methodological quality and reporting transparency persist. Diagnostic testing during the recent pandemic brought those concerns into the public domain. The Minimum Information for Publication of Quantitative PCR Experiments (MIQE) guidelines, introduced in 2009 and updated in 2025, were intended to standardize assay design and reporting, but their impact has been modest.

We assessed trends in RT-qPCR methodological reporting between 2007 and 2025 using PubMed Central searches and manual evaluation of 355 full-text articles from 2019 and 2024. Parameters analyzed included RNA integrity assessment, oligonucleotide sequence disclosure, reference gene validation, PCR efficiency reporting, and MIQE citation. In addition, targeted cohorts of 50 "reference gene" and 50 "PCR efficiency" publications from 2024/25 were evaluated. Results were compared across timepoints, geographic regions, and MIQE-citing vs non-citing studies.

Reporting of core parameters remained low or declined. Between 2019 and 2024, RNA integrity reporting fell (22% to 11%), reference gene validation was rare (13% to 5%), and PCR efficiency reporting collapsed (13% to 1%). MIQE-citing papers in 2024 showed better adherence (31% RNA integrity, 47% reference gene validation, and 40% PCR efficiency) but still omitted essential details. Asia now dominates RT-qPCR output by volume, while Europe contributes most MIQE-citing studies. Targeted cohorts reported more methodological information, yet many still failed to meet basic standards. These findings confirm that incomplete experimental design and reporting continue to undermine reproducibility and robustness of RT-qPCR assays.