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Page 14 of 19 Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69

The separation of blood into cellular and liquid layers in the presence of an anticoagulant through
centrifugation allows for the isolation of plasma. Blood plasma provides a rich source of miRNAs. It offers a
non-invasive and easily accessible sample type, collected through standard venipuncture techniques, which
makes it convenient for use in diagnostic testing. Moreover, plasma is stable, abundant, and compatible
with this protocol. Additionally, the systemic representation of plasma allows for longitudinal monitoring,
providing broader insights into overall health status. While miRNAs may be recovered from serum, the
number of recovered miRNAs will differ significantly. For example, a recent study comparing miRNA
recovered from plasma vs serum of acute myocardial infarction patients revealed differential expression
patterns . Therefore, we recommend the use of plasma for this protocol.
[18]

During the plasma isolation process, it is important to monitor for hemolysis as it likely indicates sample
[19]
degradation or mishandling . Use of a large bore (e.g., 18 gauge) collection needle, and gentle handling
during processing can limit hemolysis. Hemolysis can compromise the accuracy, specificity, and reliability
of results due to the presence of PCR inhibitors, contamination with genomic DNA, and distortion of target
nucleic acid concentration . Hemolytic samples should be excluded from analysis.
[19]
Proper handling and transfer of plasma to sterile, nuclease-free tubes ensure preservation for quantitative
ddPCR analysis. Snap freezing rapidly preserves samples, typically using liquid nitrogen or a slurry or dry
[20]
ice and isopropanol . This method must be performed when preserving plasma and prior to downstream
storage of isolated nucleic acids, as it helps to prevent the formation of large ice crystals, which can damage
the nucleic acids. Cryo-preservation of plasma at temperatures of -80 °C or below helps inhibit enzymatic
activity and chemical reactions that lead to nucleic acid degradation. To maintain miRNA integrity during
storage and handling, it is important to limit freeze-thaw cycles. Each cycle can cause significant nucleic
acid degradation or fragmentation. Storing samples in aliquots eliminates the negative effects of freeze-thaw
cycles.

During the freeze-thaw process, nucleic acids are vulnerable to damage from changes in pH, physical
shearing, and denaturation . Slowly thawing samples minimizes damage by allowing for gradual pH
[21]
change, reducing the rate of ice crystal formation, and allowing the nucleic acids to refold, reducing
denaturation. Using sterile, nuclease/pyrogen-free water/buffers/tubes, freezing the sample rapidly, thawing
them slowly, and avoiding repeated freeze-thaw cycles are all essential components of reproducibility.

There are myriad ways to isolate miRNAs from plasma, each with its own capacities, advantages,
disadvantages, and idiosyncrasies. For example, we have found that the phenol-chloroform extraction
method is incredibly cost-effective and efficient for the isolation of messenger (mRNAs) [13,22] . However, this
method does not efficiently isolate small RNAs from plasma. Adding different components, such as
glycogen, can mitigate this to a degree, but because circulating miRNA levels are relatively low, these
methodologies cannot yield enough for direct quantification by this method. While pre-amplification has
been used in the past, these additional modifications result in inevitable variations, making results less
comparable across studies.

For this protocol, we have chosen the miRNeasy Serum/Plasma Advanced Kit from Qiagen, which has
[9]
previously been reported to be among the optimal kits to isolate miRNA from plasma . The kit utilizes a
silica-based membrane and chaotropic salt to selectively bind and elute small RNAs. Although it is not the
most cost-efficient method available, it is comprehensively effective, standardized, and can be automated.