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MicroRNAs

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...

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Challenges in circulating miRNA analysis in adrenocortical tumors.

Bálint Vékony, Gábor Nyirő, Henriett Butz

Endocrine-Related Cancer

|June 5, 2025

Summary

Digital PCR (dPCR) and quantitative PCR (qPCR) are not interchangeable for measuring circulating microRNAs (miRNAs) due to method-specific biases and anticoagulant effects. Standardized protocols are crucial for reliable adrenocortical cancer diagnostics.

Keywords:

EDTA adrenocortical carcinoma dPCR microRNA qPCR

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Area of Science:

Endocrinology
Molecular Biology
Biomarker Discovery

Background:

Adrenocortical tumors require accurate differentiation between benign and malignant types.
Circulating microRNAs (miRNAs) show potential as biomarkers for adrenocortical cancer (ACC).
Technical and biological standardization challenges hinder miRNA biomarker utility.

Purpose of the Study:

To evaluate the interchangeability of quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) for circulating miRNA measurement.
To investigate the influence of K2-EDTA and K3-EDTA anticoagulants on miRNA quantification.
To assess method-specific biases and anticoagulant effects on miRNA biomarker accuracy.

Main Methods:

Simultaneous blood collection from 20 participants into K2-EDTA and K3-EDTA tubes.
Analysis of three ACC-associated miRNAs (miR-483-5p, miR-210-3p, miR-21-5p) and two controls (miR-16-5p, cel-miR-39-3p).
Measurement using both RT-qPCR and dPCR techniques.

Main Results:

qPCR and dPCR yielded different correlations, with K2-EDTA showing better performance in ΔCt values.
Proportional biases were observed between methods, particularly for low or high miRNA expressions.
K3-EDTA influenced qPCR measurements, increasing standard deviations, while dPCR results were unaffected by anticoagulant choice.

Conclusions:

dPCR and qPCR are not readily interchangeable for circulating miRNA analysis, impacting cross-validation studies.
Anticoagulant choice (K2- vs. K3-EDTA) can affect qPCR outcomes, necessitating standardized protocols.
A consensus methodology is essential to improve reproducibility and enhance miRNA biomarker utility in adrenocortical tumor diagnostics.