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INTRODUCTION
MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in regulating
translation. They are secreted by most cell types and involved in a variety of physiological and pathological
processes. Moreover, recent studies demonstrate that miRNAs are promising potential biomarkers for
[1,2]
disease diagnosis, prognosis, and therapy response . Circulating miRNAs - that is, those found in plasma -
are of particular interest because they are relatively stable and easily accessible. Moreover, we have
established that patients with thoracic aortic aneurysms have a unique plasma miRNA profile, which has
[3]
demonstrated exceptional sensitivity and specificity as a diagnostic tool .

However, quantifying miRNAs in plasma is challenging for several reasons, chief among them being a lack
of reproducible, standardized protocols. Because there are many ways to perform each step of this protocol,
results are often not comparable between studies. The foundation of this protocol is the standardization of
blood collection and storage, miRNA isolation and normalization, and multiplexed target quantification. As
such, the use of this protocol will facilitate multi-study comparison and allow systematic review and meta-
analyses.

Digital droplet PCR (ddPCR) has immense potential and is a viable tool with which to overcome the many
challenges intrinsic to measuring miRNAs in plasma. Herein, we present a validated method for the
absolute quantification of circulating, cell-free miRNA utilizing miRNA-specific primers and DNA binding
dyes measured by ddPCR. We employ two normalization steps: firstly, the input amount of miRNA is
standardized, and secondly, we utilize an exogenous miRNA (miR-39) spike-in to normalize the cDNA
generation step. ddPCR relies on the dilution and partitioning of a sample into thousands of individual
droplets. Some will contain the target of interest, while others do not. PCR amplifies fluorescence intensity
only in the droplets containing the targets of interest. A droplet reader then counts each droplet to
determine the number of positive and negative values for each miRNA. This process allows for the absolute
quantification of the copy number of a target miRNA. Previous studies have demonstrated that ddPCR
quantification of miRNA results in much greater precision of values obtained compared to real-time PCR,
in addition to absolute quantification . Several previous studies have also applied ddPCR for the
[4]
quantification of circulating cell-free miRNA .
[4-8]
This technical note delineates the workflow for the quantification of miRNAs in its entirety: we begin with
sample collection/preparation, reverse transcription, droplet generation, PCR amplification, and finish with
data analysis. We have also provided insights and guidance regarding optimization, assay conditions, and
the troubleshooting of common issues. Stepwise normalization and detailed workflow allow for
reproducibility. It can easily be adapted to quantify most circulating miRNAs in plasma, making it a
valuable resource for diagnostic development.

PROTOCOL
1. Preparation of miR-39 exogenous spike-in [Figure 1].

A stepwise protocol is available in the [Supplementary File 1].

Resuspension and serial dilution of miR-39 exogenous spike-in
The first step in this workflow is to prepare the exogenous spike-in, cel-miR-39-3p. Normally, miR-39
mimic oligonucleotides are supplied in lyophilized form following standard purification at either 5 or
20 nmol (mirVana® miRNA mimic Assay ID: MC10956; miRbase v22.1 Accession Number: MI0000010).
The following instructions and corresponding Figure 1A detail the preparation of the spike-in from a tube