Circulating Cell-Free RNA Reflects Immune-Associated and Airway Remodeling Signatures in Equine Asthma

We profiled cfRNA from 16 EA cases (including mastocytic, neutrophilic, and paucigranulocytic phenotypes) and 12 healthy controls using cfRNA-stabilized blood collection and UMI-based total RNA-seq optimized for low-input samples. For comparative molecular profiling, we sequenced bronchoalveolar lavage (BAL) fluid RNA (n=12: 7 EA, 5 controls) and whole blood RNA (n=11: 6 EA, 5 controls) from partially matched individuals, with 9 horses providing complete three-compartment sampling.

RNA-seq libraries were prepared using the SMARTer Stranded Total RNA-Seq Pico Input Kit with UMIs (Takara Bio, v3/v4) and sequenced as paired-end 150 bp reads on an Illumina NovaSeq X platform. Our analysis revealed that cfRNA captures biologically relevant inflammatory and airway remodeling signatures characteristic of asthma, including altered immune cell composition and upregulation of key alarmins and remodeling-associated transcripts.