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Transcriptional changes associated with equine recurrent airway obstruction (RAO)

We determined differentially expressed genes using differential display techniques in horses with naturally acquired recurrent airway obstruction (RAO) and compared them with those of clinically healthy horses. Standard extraction techniques with Tri‐Reagent were used to isolate total RNA from frozen lung tissue from 6 horses with RAO and 6 healthy horses. The differential display PCR was conducted with GeneFishing DEGs 103 to 110 systems following the manufacturer's instructions. The differentially expressed bands were excised from the gel, purified, and were cloned into Invitrogen pTOPO TA vector. At least 3 colonies resulting from each transformation were analyzed for recombinant plasmids by PCR with universal primers. The recombinant colonies had the plasmids extracted and purified DNA was sequenced. There were 88 differentially expressed genes identified after blasting against the NCBI database. Of these, 70 were up‐regulated and 18 were down‐regulated. Validation was performed by RT‐PCR assay on four selected up‐regulated genes: uridine phosphorylase 1 (metabolic intermediate biosynthesis), annexin A2 (function), beta 2 microglobulin (function) and thymosin beta 4 (function). These results demonstrate the complexity of RAO and contribute to a better understanding of molecular mechanisms responsible for the progression of this devastating disease.