A high-throughput, end-to-end pipeline for extracellular miRNA biomarker discovery from human biofluids

We developed a scalable pipeline for extracellular miRNA (ex-miRNA) profiling that integrates automated exRNA extraction, small RNA sequencing, and bioinformatic analysis including data processing and normalization. Automated extraction protocols, including doubling input volume or lyophilization to increase RNA yield, were benchmarked against leading manual methods, with donor pregnancy status serving as the primary biological variable. Small RNA sequencing was performed across conditions, enabling systematic evaluation of data quality and comparison of normalization strategies. Across methods and biofluids, DESeq2 most effectively reduced technical variability while preserving biological signal. Comparing specimen types, plasma exhibited the highest reproducibility and retention of biological signal, followed by serum, while urine exhibited greater variability and less differentially expressed miRNAs. Pregnancy-associated ex-miRNA signatures, including C19MC miRNAs, were consistently detected in both plasma and serum. Together, this study establishes a robust framework for scalable exRNA extraction and profiling, supporting standardized assay development for biomarker discovery and clinical applications.