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Maiocchi et al. Vessel Plus 2023;7:27 Vessel Plus
DOI: 10.20517/2574-1209.2023.69

Technical Note Open Access

Plasma microrna quantification protocol

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Sophie Maiocchi , Elizabeth N. Collins , Andrew R. Peterson , Kyle C. Alexander , Dalton J. McGlamery ,
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Noah A. Cassidy , John S. Ikonomidis , Adam W. Akerman 2
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Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.
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Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-
7065, USA.
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University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Correspondence to: Asst. Prof. Adam W. Akerman, PhD, Department of Surgery, Division of Cardiothoracic Surgery, University
of North Carolina at Chapel Hill, MBRB 111 Mason Farm Road, Suite 6340, Chapel Hill, NC 27599 USA. E-mail:
adam_akerman@med.unc.edu
How to cite this article: Maiocchi S, Collins EN, Peterson AR, Alexander KC, McGlamery DJ, Cassidy NA, Ikonomidis JS,
Akerman AW. Plasma microrna quantification protocol. Vessel Plus 2023;7:27. https://dx.doi.org/10.20517/2574-1209.2023.69
Received: 15 Jun 2023 First Decision: 25 Sep 2023 Revised: 18 Oct 2023 Accepted: 9 Nov 2023 Published: 16 Nov 2023
Academic Editor: Narasimham L. Parinandi Copy Editor: Fangling Lan Production Editor: Fangling Lan

Abstract
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many
pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic
aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized
reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a
thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive
protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood
collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR
amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization,
assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise
normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this
protocol may be automated. The data provided in this technical note is demonstrative of the values typically
obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected
aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to
quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Keywords: MicroRNA, plasma, quantification, aortic aneurysm, ddPCR

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made.

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