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to fully exploit the diagnostic potential of plasma miRNAs and ddPCR in clinical settings.

The accurate quantification of miRNAs from blood samples is a crucial aspect of molecular diagnostics and
research. By following standardized protocols for blood collection, preservation, and processing, researchers
can ensure the integrity and stability of nucleic acids, which is essential for reliable PCR analysis.
Standardizing the use of anticoagulants, minimizing freeze-thaw cycles, and incorporating spike-in controls
are critical steps in achieving accurate and reproducible miRNA quantification. Furthermore, the utilization
of advanced techniques such as loop-specific cDNA generation and droplet partitioning through digital
PCR provides enhanced sensitivity, precision, and multiplexing capabilities for miRNA analyses. By
implementing these methodologies and adhering to rigorous quality control measures, researchers can
overcome technical limitations, enhance the accuracy of miRNA quantification, and pave the way for
improved diagnostics.

DECLARATIONS
Acknowledgments
Graphic design by Jeongeun Annie Kim.

Authors’ contributions
Conceptualization; writing - original draft: Ikonomidis JS, Alexander KC, Maiocchi S, Akerman AW
Data curation: Cassidy NA, Peterson AR, Collins EN, Maiocchi S, Akerman AW
Formal analysis: Ikonomidis JS, Cassidy NA, Collins EN, Maiocchi S, Akerman AW
Funding acquisition: Ikonomidis JS, Akerman AW
Investigation: Maiocchi S, Akerman AW
Methodology: Cassidy NA, McGlamery DJ, Peterson AR, Collins EN, Maiocchi S, Akerman AW
Project administration; supervision: Akerman AW
Resources: Alexander KC, Akerman AW
Software: Alexander KC, Collins EN, Maiocchi S, Akerman AW
Validation: McGlamery DJ, Collins EN, Maiocchi S, Akerman AW
Visualization: Alexander KC, Maiocchi S, Akerman AW
Writing - review & editing: Ikonomidis JS, Cassidy NA, Alexander KC, Collins EN, Maiocchi S, Akerman
AW

Availability of data and materials
All relevant data are within the manuscript and its Supplementary Materials.

Financial support and sponsorship
Research reported in this publication was supported by the University of North Carolina at Chapel Hill and
the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health: R01HL169390
(PI: Akerman AW); R56HL161454-01A1, and R21HL148363 (PI: Ikonomidis JS); 2021 UNC Idea Grant:
Development of a Liquid Biopsy Based Diagnostic Test for Assessing Thoracic Aortic Aneurysm Disease
(PI: Akerman AW). The content is solely the responsibility of the authors and does not necessarily represent
the official views of UNC or the National Institutes of Health.

Conflicts of interest
All authors declared that there are no conflicts of interest.