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Page 16 of 19 Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69

[27]
Several small RNAs, like miR-16 and RNU6B, have been identified to be stable in specific pathologies .
Unlike miRNAs, small-nucleolar RNAs, like RNU6B, are subject to entirely different regulatory processes,
making normalization less relevant. Nevertheless, none of the proposed small RNA housekeeping molecules
have been demonstrated to be stably expressed across cardiovascular diseases/co-morbidities. This problem
is multiplied when one considers complex pathologies with many subtypes and genetic preconditions, such
as aortic aneurysm disease, making it easy to see that previous methods are not feasible in this context.

To avoid the issues described above, we developed a method for normalization to a miRNA spike-in using
the synthetic, exogenous nucleic acid molecule miR-39. It is prepared and validated prior to being added to
samples to serve as a reference for data normalization and control for inevitable variations in all
experiments. These controls help monitor experimental efficiency and accuracy and allow for the
normalization of gene expression levels between specimens.

The specificity of target amplification is enhanced when loop-specific cDNA generation is used, as in
TaqMan miRNA assays, to convert miRNAs into complementary DNA (cDNA) for subsequent PCR
amplification. The chemistry involves a small-RNA specific, stem-looped oligonucleotide primer that
specifically hybridizes to the 3' end of each target miRNA. The resulting cDNA is then amplified and
quantified using TaqMan PCR primers and probes.

Hydrolysis probe chemistry is another essential component of miRNA quantification. Hydrolysis probes
bind to the target miRNA sequence and have a fluorescent reporter dye at one end and a quencher molecule
at the other. During PCR amplification, the probe binds specifically to the target miRNA sequence, and
when the RNA polymerase reaches the probe during the amplification process, it separates the reporter dye
from the quencher. This reaction is observed as a fluorescent signal, which is detected and quantified in
each droplet of the ddPCR system.

When performing this protocol, it is important to adhere to the most recent ddPCR MIQE guidelines . In
[28]
accordance with these guidelines, all TaqMan small RNA assays have been tested and optimized for
specificity, reproducibility, linear dynamic range, sensitivity, and efficiency, which are all available via
ThermoFisher. This enhances overall reproducibility and consistency when comparing analyses across
laboratories.

Droplet generation is a critical step in this workflow. Microfluidics technology partitions the PCR reaction
into thousands of droplets, each acting as a separate reaction chamber. This enables the amplification and
quantification of miRNAs with high sensitivity and accuracy.

Plasma miRNAs hold great promise as potential diagnostic markers due to their stability, ease of collection,
and association with various diseases. However, there are certain limitations that need to be considered.
First, the presence of abundant non-specific miRNAs and other extracellular RNA species in plasma can
hinder the specificity and sensitivity of miRNA detection. This necessitates the use of stringent purification
methods and the inclusion of appropriate controls to minimize false-positive results. Additionally, the
variability in miRNA expression patterns among individuals and the lack of standardized reference genes
make it challenging to establish universally applicable diagnostic thresholds. The use of an exogenous spike-
in control mitigates this challenge. Furthermore, while ddPCR offers enhanced sensitivity and precision, it is
limited by the number of miRNA targets that can be simultaneously analyzed in a single reaction due to the
partitioning of the sample into droplets. Overcoming these challenges requires further optimization of
purification techniques, standardization of reference genes, and the development of multiplexing strategies