Comprehensive profiling of the human tear fluid miRNome using small RNA sequencing

Purpose: To generate a comprehensive profile of microRNAs (miRNAs) present in human tear fluid using next-generation sequencing (NGS), establish a reference miRNome for healthy human tear fluid, and investigate whether miRNA expression varies by sex, race, or age. Methods: Tear samples were collected from 32 adults using Schirmer strips. RNA was isolated using the miRNeasy Serum/Plasma Kit, and cDNA libraries were prepared using the QIAseq miRNA Library Kit. Barcoded libraries were sequenced on the NovaSeq 6000 platform. Bioinformatic analyses included adapter trimming, alignment to miRBase, normalization with DESeq2, and functional annotation using multiMiR and clusterProfiler. Differential expression analysis was performed to evaluate variability across demographic groups. Results: We identified 318 miRNAs constitutively expressed in tear fluid, 246 of which were detected in all subjects. The most abundant miRNAs included hsa-let-7a-5p, hsa-let-7f-5p, hsa-let-7b-5p, hsa-let-7g-5p, and hsa-let-7c-5p. Prominent miRNA families included let-7 (13 members), miR-30 (10), miR-8 (10), miR-10 (8), and miR-17 (8). Functional enrichment analyses indicated that tear miRNAs may regulate pathways involved in transcriptional control, cytoskeletal organization, MAPK signaling, and neurodegenerative processes. Samples processed in two batches several months apart showed strong concordance, confirming methodological reproducibility. Two miRNAs, hsa-miR-375-3p and hsa-miR-708-5p, were significantly increased in White subjects, whereas hsa-miR-106b-3p and hsa-miR-205-3p showed a nonsignificant increase in females. Conclusions: This study defines a high-resolution reference profile of the human tear fluid miRNome, demonstrating its stability, diversity, and functional relevance. These data establish a robust baseline for future studies investigating disease-associated alterations in tear miRNAs. Observed demographic associations remain exploratory and require larger, adequately powered cohorts.