Challenges in circulating miRNA analysis in adrenocortical tumors

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 anticoagulants 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 related to low or high miRNA expressions 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 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.