Messenger RNA from Canine Cytology Lymph Node Specimens Can Be Utilised for Gene Expression Profiling Using Digital RNA Counting

Gene expression profiling (GEP) is being used with increasing frequency to characterise canine tumours, but canine samples are often not collected in a medium to preserve RNA. Cytology samples from human tumours have been shown to provide adequate RNA for GEP using NanoString nCounter technology, suggesting a similar approach might be viable for canine samples. Determine the quantity and quality of RNA extracted from canine cytology slides, evaluate the time frame for RNA integrity and assess the utility of performing GEP with extracted RNA. Cytological specimens were derived from reactive lymph nodes (n = 10) and lymphoma cases (n = 34) and characterised via flow cytometry or PCR for antigen receptor rearrangements. RNA was extracted from stained and unstained lymph node cytology slides using a RNeasy Mini Kit and subsequently evaluated for quality and quantity. GEP was carried out using nCounter technology with the NanoString Canine IO Panel. Extracted RNA from lymphoma and reactive cytology samples generated concentrations ranging from 27.5 to 543.9 ng/μL (median: 168.5 ng/μL; interquartile range (IQR): 108.15-275.95 ng/μL). RNA integrity numbers (RINs) ranged from 1.0 to 7.2 (median: 2.9; IQR: 2.10-4.45) and DV200 values ranged from 50.5% to 93.2% (median: 81.65%; IQR: 71.95%-87.85%). RIN and DV200 values were highest for extractions within 7 days of sample collection. GEP revealed overexpression of 17 genes in lymphoma samples such as MYC, TERT and BRCA2. Canine lymph node cytologic specimens provide sufficient RNA quality for GEP. The nCounter Canine IO Panel demonstrates the utility of cytology specimens for evaluating gene expression.