Consensus guidelines for the validation of qRT-PCR assays in clinical research by the CardioRNA consortium

David de Gonzalo-Calvo, Monica Marchese, Jan Hellemans, Fay Betsou, Nanna Lond Skov Frisk, Louise Torp Dalgaard, Päivi Lakkisto, Carole Foy, Andreas Scherer, María Laura Garcia Bermejo, Yvan Devaux*, EU-CardioRNA COST Action CA17129 (

*Corresponding author for this work

Research output: Contribution to journalReview articlepeer-review

11 Citations (Scopus)


Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking. Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and in vitro diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium ( Their applicability encompasses all clinical areas.

Original languageEnglish
Pages (from-to)171-180
Number of pages10
JournalMolecular Therapy - Methods and Clinical Development
Publication statusPublished - 10 Mar 2022


  • assay validation
  • clinical research assays
  • noncoding RNA
  • PCR
  • RNA


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