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Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69 Page 15 of 19

The importance of beginning analysis with even amounts of isolated miRNA cannot be overstated. Isolation
efficiency depends largely upon handling and the presence of contaminants. There are several different ways
to measure and normalize the amount of total miRNA. These include volume corrections following analysis
by spectroscopy, electrophoresis, and/or nucleic acid dyes (as in the Qubit Assay).

The Invitrogen Qubit miRNA assay utilizes non-sequence specific nucleic acid dyes that emit fluorescence
only when bound to miRNA (17-25 bp), offering an accurate and selective method for quantification. The
fluorescence signal is proportional to the amount of miRNA. Samples are compared to a standard curve,
making it a direct quantification rather than a surrogate measure as with spectroscopy. The dye is highly
selective for intact miRNA over ribosomal (rRNA) or larger mRNAs (> 1,000 bp). The kit provides accurate
results for miRNA concentrations ranging from 0.025 to 150 ng/µL, with a detection range of 0.5-150 ng in
volumes between 1 µL and 20 µL. Furthermore, fluorescence measurements are more sensitive, allowing for
reduced noise levels. In comparison to traditional UV absorbance methods, this approach offers improved
accuracy by mitigating overestimations caused by contaminants such as salts, solvents, detergents, proteins,
and free nucleotides .
[23]
Multiple studies have compared different quantification methods of total miRNA [24,25] . Garcia-Elias and
colleagues directly compared the Infinite® 200 PRO Nanoquant and Nanodrop 2000 spectrophotometers,
and the Agilent 2100 Bioanalyzer PicoChip and SmallChip, and the Qubit® 2.0 Fluorometer. Their results
showed that the spectrophotometric measurements overestimated RNA concentration due to the detection
of contaminants. These results are consistent with those of Wright et al., who also found that the Nanodrop
consistently reported higher concentrations of RNA compared to the BioAnalyzer and the Qubit. Thus, we
strongly recommend the use of the Qubit, where available.

RNA Integrity Number (RIN) is a score that measures the intactness of isolated RNA molecules -intactness,
in this context, is commonly called integrity or quality. RIN was developed as a quality assessment metric
[25]
based on nucleic acid length . The RIN value is based on the electrophoretic profile of RNA from cells or
tissue, which contain classes of ribosomal RNA such as 18 s and 28 s rRNA. These are used for the RIN
algorithm and can be problematic in this application for several reasons. First, plasma and serum have
inherently lower populations of 18s and 28s rRNA and so typically provide lower RIN values in this
context . Second, isolated miRNA are enriched for small RNA (< 25 bp), so the RIN value is irrelevant. For
[25]
these reasons, RIN cannot be reliably used to determine small RNA quality. The Qubit miRNA assay
specifically targets a narrow and specific nucleic acid size-length range. In this protocol, if measured values
fell outside the range reported in Figure 3, results likely indicated degradation or contamination. We suggest
that compromised samples with concentrations outside these ranges must be excluded from subsequent
analysis.

While standardization of starting input is indispensable, inherent constraints and natural variability
necessitate additional corrections to account for differences in reaction efficiencies and overall
quantification. Traditionally, internal referent normalization accounts for these variations. It involves
comparing target values to a reference gene. The ideal reference gene should be unaffected by the
experimental treatment or pathology and should be expressed at a similar level to that of the target to
preserve the linear dynamic range of detection. Several “housekeeping” mRNAs are generally accepted and
widely used (e.g., GAPDH, beta-actin) . The former cannot function in this protocol, as quantification
[26]
would require different chemistries.