Development of circulating microRNA-based biomarkers for medical decision-making: a friendly reminder of what should NOT be done

Abstract

Circulating cell-free microRNAs (miRNAs) represent a major reservoir for biomarker discovery. Unfortunately, their implementation in clinical practice is limited due to a profound lack of reproducibility. The great technical variability linked to major pre-analytical and analytical caveats makes the interpretation of circulating cell-free miRNA data challenging and leads to inconsistent findings. Additional efforts directed to standardization are fundamental. Several well-established protocols are currently used by independent groups worldwide. Nonetheless, there are some specific aspects in specimen collection and processing, sample handling, miRNA quantification, and data analysis that should be considered to ensure reproducibility of results. Here, we have addressed this challenge using an alternative approach. We have highlighted and discussed common pitfalls that negatively impact the robustness of circulating miRNA quantification and their application for clinical decision-making. Furthermore, we provide a checklist usable by investigators to facilitate and ensure the control of the whole miRNA quantification and analytical process. We expect that these recommendations improve the reproducibility of findings, and ultimately, facilitate the incorporation of circulating miRNA profiles into clinical practice as the next generation of disease biomarkers.

Keywords:

• Biomarker
• microRNA
• limitation
• methodology
• pitfall

Acknowledgments

We would like to thank all EU-CardioRNA COST Action members for insightful discussions that contributed to drafting the current paper.

Disclosure statement

DdGC and YD holds patents related to diagnostic and therapeutic applications of RNAs.

Additional information

Funding

This article is based upon work from EU-CardioRNA COST Action CA17129 (https://cardiorna.eu/) supported by COST (European Cooperation in Science and Technology). DdGC has received financial support from Instituto de Salud Carlos III [Miguel Servet 2020: CP20/00041], co-funded by the European Social Fund (ESF)/“Investing in your future.” This work is supported by Instituto de Salud Carlos III [PI20/00577], co-funded by European Regional Development Fund (ERDF)/“A way to make Europe.” CIBERES is an initiative of the Instituto de Salud Carlos III. YD is funded by the EU Horizon 2020 project COVIRNA (Grant Agreement # 101016072), the National Research Fund [grants # C14/BM/8225223, C17/BM/11613033, and COVID-19/2020-1/14719577/miRCOVID], the Ministry of Higher Education and Research, and the Heart Foundation-Daniel Wagner of Luxembourg. PL is funded by the Finnish Cultural Foundation, The Finnish Foundation for Cardiovascular Research, The Finnish Society of Clinical Chemistry, and the Finnish Foundation for Laboratory Medicine.