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<p><em>Background:</em> UC is the most common urogenital cancer, comprising up to 2% of all naturally occurring neoplasia in dogs and can be challenging to diagnose. With early diagnosis, the disease can be controlled in most dogs with a good quality of life. MiRNAs are small non-coding RNAs that function by post-transcriptional regulation of gene expression. Their abundant presence and stability in the body make them promising tools for disease diagnosis. </p> <p><em>Hypothesis:</em> A microRNA (miRNA) signature can be used to differentiate canine urothelial carcinoma (UC) from other lower urinary tract diseases.</p> <p><em>Literature review:</em> There is an overlap of miRNA expression changes between normal physiologic processes, non-infectious and non-inflammatory conditions, infectious and/or inflammatory conditions, and neoplasia. Additionally, the mechanism of action of these overlapping miRNAs varies depending on the disease process. There is a lack of standardization of miRNA evaluation and consistency within a single evaluation method. Herein we evaluate three papers on miRNA expression in canine UC and compared the reported expression profile to human UC literature and identified experimentally validated targets of the dysregulated miRNA. </p> <p><em>Methods and results:</em> <strong>(Aim 1)</strong> Using reverse transcriptase quantitative PCR (RT-qPCR), we assessed the effects of sample handling on miRNA expression in formalin-fixed Paraffin-embedded (FFPE) tissue and urine sediment. We showed that the time of tissue fixation in formalin does not alter the detection of miRNA expression, but the inclusion of the muscularis layer altered the miRNA expression profile in bladder tissue. Additionally, miRNAs in urine sediment were proven to be stable despite the storage temperature for up to two weeks. <strong>(Aim 2)</strong> Using Next Generation Sequencing (NGS) with validation of findings via RT-qPCR, we evaluated differential miRNA expression in bladder tissue collected from normal canine urothelium and the invasive type of UC (iUC) to elucidate the dysregulated pathways. We found that twenty-eight miRNAs were differentially expressed (DE). The DE miRNAs were most often associated with gene silencing by miRNA, miRNAs in cancer, and miRNAs involved in DNA damage responses. Proteins involved include HRAS, KRAS, ARAF, RAF1, MAPK1, MAP2K1, MAPK3, FGFR3, EGFR, HBEGF, RASSF1, E2F2, E2F3, ERBB2, SRC, MMP1, and UP3KA. <strong>(Aim 3)</strong> Using RT-qPCR, expression of miR-214, miR-181a, miR-361, and miR-145 were evaluated. We failed to reject the null hypothesis that the relative gene expression in all groups was the same for any miRNA, nor did we find any multivariate summary that could effectively differentiate UC from inflammatory and non-neoplastic transitional cells. </p> <p><em>Conclusions:</em>   The findings within this thesis highlight the need for standardized methods for miRNA evaluation, support the use of stored samples for miRNA expression analysis, and show the importance of isolating the tissue of interest in FFPE. We defined the miRNome of iUC and investigated numerous protein pathways affected by dysregulation of differentially expressed miRNA in urothelial carcinoma. While we failed to reject our null hypothesis that the miRNA signature we evaluated could be utilized as a diagnostic tool for canine urothelial carcinoma, we showed the promise of miRNA as diagnostic tools and highlight several novel pathways that miRNA regulation affects in this disease. </p>