Evaluation of fecal microRNA stability in healthy cats

Abstract

BackgroundGastrointestinal (GI) cancer accounts for 14% of feline malignancies. There is a great need for reliable noninvasive diagnostic biomarkers to reach a timely diagnosis and initiate treatment. Fecal microRNAs (miRNAs) could be such a biomarker and have shown great potential in colorectal screening in people but have yet to be investigated in cats.ObjectivesWe aimed to evaluate the presence and stability of feline fecal miRNA under different storage conditions (room temperature [RT], 4, and −20°C) and to evaluate the expression levels of specific fecal miRNAs collected on three separate days (days 1, 4, and 7) in healthy cats.MethodsHealthy cats were prospectively recruited. Fecal samples were collected, aliquoted, and stored for 24 hours at RT and then transferred to −20°C, stored for 24 hours at 4°C and then transferred to −20°C, or were immediately placed at −20°C on day 1 or at −20°C on days 4 and 7 postcollection. Expression of 22 miRNAs was investigated using quantitative real‐time PCR.ResultsTen miRNA assays worked well, and one, let‐7b, was used for normalization. No differences in miRNA expression were seen between the three storage temperatures for the nine miRNAs investigated. Only miR‐26a showed a significant increase in expression between samples of days 1 and 7. The rest of the miRNAs levels were stable over time.ConclusionsFecal miRNA can be isolated from healthy cats. The expression was stable at different temperatures and for most of the miRNAs over time. Prospective studies evaluating fecal miRNA as biomarkers in cats with GI neoplasia are warranted.

Association of fecal and serum microRNA profiles with gastrointestinal cancer and chronic inflammatory enteropathy in dogs

“…Fecal samples were collected and aliquoted (approximately 1 g per aliquot) into cryotubes within 1 hour of defecation and stored at −20°C for miRNA quantification. This was done at the presentation or at home by the client after receiving thorough verbal and written instructions and the necessary supplies as previously described 15,28 . Mucosal endoscopic pinch biopsies or surgical full‐thickness biopsies were obtained at the clinician's discretion.…”

“…For fecal samples, total RNA was isolated from 100 mg fecal material using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) as described previously 28 …”

Association of serum and fecal microRNA profiles in cats with gastrointestinal cancer and chronic inflammatory enteropathy

“…The miRNeasy Mini Kit (Qiagen, Hilden, Germany) was used for isolation of total RNA from 100 mg feces as described previously 14 . Total RNA was isolated from 200 μL serum using the miRNeasy Micro Kit (Qiagen, Hilden, Germany) without including the accompanying C. elegans miR‐39 spike‐in extraction control.…”

“…The qPCR was carried out using the high‐throughput platform Biomark HD (Fluidigm, San Francisco, California) and the 96.96 Dynamic Array integrated fluidic circuit chip format (Fluidigm, San Francisco, California). All details on RT‐qPCR analysis can be found in File S1 and in Table S1 12‐14,19‐47 …”

“…10,11 Likewise, several miRNAs are abundant in GIC and chronic enteropathies in dogs. 12,13 MiRNAs already have been successfully measured in feces from healthy cats, 14 but their potential as noninvasive diagnostic biomarkers in serum and feces for differentiation between GIC and CIE in cats remains to be investigated.…”