Digital PCR analysis of ALS plasma miRNA biomarkers: towards analytical consistency and pathogenic relevance

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by motor neuron degeneration without properly standardized molecular biomarkers for early diagnosis or disease monitoring. Diagnosis remains primarily clinical, often involving lengthy exclusion processes and significant delays, highlighting the need for minimally invasive, accessible, and disease-specific molecular tools. Circulating microRNAs (miRNAs) are promising biomarker candidates due to their stability in plasma/serum and roles in gene regulation. This study aimed to quantify the expression of selected ALS-associated plasma miRNAs using digital PCR (dPCR) and compare them against those in healthy controls to validate their efficacy as early ALS biomarkers. Plasma samples were collected from eight ALS patients and four age-and-sex-matched healthy controls. Four candidate target miRNAs (hsa-miR-93-5p, hsa-miR-181a-5p, hsa-miR-206, hsa-miR-7111-3p) and one endogenous control (hsa-miR-191-5p) were selected via systematic review. Total miRNA concentration did not significantly differ between ALS patients and controls, suggesting global miRNA levels are not altered in ALS. Hsa-miR-206 was consistently upregulated across all analysis strategies, supporting its role as a robust biomarker. Hsa-miR-93-5p also showed significant differential expression, though results were dependent on normalization approach. Correlation analysis with ALS Functional Rating Scale-Revised (ALSFRS-R) scores revealed moderate but non-significant trends for both miRNAs. Target prediction analysis linked both miRNAs to ALS-associated genes, suggesting potential involvement in disease pathways. The study concludes that miR-206 is a promising candidate for clinical translation as a diagnostic and prognostic biomarker. It also emphasizes the importance of methodological standardization in miRNA research to ensure reproducibility and clinical applicability. Further validation in larger cohorts is necessary to confirm these findings.