The differentiation of benign and malignant adrenocortical tumors is of major clinical
relevance. Circulating microRNAs (miRNAs), hold promise as blood-borne biomarkers
of adrenocortical cancer (ACC). There are, however, many difficulties with their use
including technical and biological standardization challenges. Our aim was to evaluate
the interchangeability of quantitative polymerase chain reaction (qPCR) and digital
PCR (dPCR) for measuring circulating miRNAs and to investigate whether K2- and K3-EDTA
as anticoagulant influence the measurements. Blood samples were drawn simultaneously
from 20 participants into K2- and K3-EDTA tubes. Three miRNAs shown to be associated
with ACC (miR-483-5p, miR-210-3p, miR-21-5p) together with two controls (miR-16-5p,
cel-miR-39-3p) were analyzed using RT-qPCR and dPCR. qPCR and dPCR results showed
different correlations in K2- and K3-EDTA samples with K2 performing better regarding
ΔCt values. Moreover, proportional biases relating to low or high miRNA expression
between the two methods were observed. In qPCR measurements, K3-EDTA samples showed
larger standard deviations, particularly for cel-miR-39. While raw Ct values differed
between K2 and K3 EDTA only for miR-483-5p, ΔCt values showed statistically significant
differences across all miRNAs except for miR-483-5p. dPCR results were not affected
by the choice of the anticoagulant. In conclusion, this is the first study demonstrating
that dPCR and qPCR results are not easily interchangeable for circulating miRNA, particularly
for abundant or rare miRNAs, making cross-validation studies challenging. K2 and K3-EDTA
could potentially influence qPCR outcomes, underscoring the need for standardized
protocols. A consensus-based methodology could improve reproducibility, enhancing
miRNA-based biomarker utility in adrenocortical tumor diagnostics.
