Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine

Abstract

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Keywords: dogs; formalin-fixation; microRNA; paraffin-embedded tissues; stability; urine; urothelial carcinoma.

Figure 1.

Histologic sections of canine urinary bladder. A. Normal urinary bladder. B. Urinary bladder with urothelial carcinoma. * = urothelium; • = lamina propria; ♦ = muscularis; ► = neoplastic urothelium (urothelial carcinoma). H&E. Image created with BioRender.

Figure 2.

Production of scroll and core tissue samples. A. Paraffin tissue block and an example of tissue scroll production. B. Tissue core sampling. FFPE-UC = formalin-fixed, paraffin-embedded–urothelial carcinoma. Image created with BioRender.

Figure 3.

Piecewise regression of microRNA in formalin-fixed tissues used to illustrate the initial effect of time on the miRNA Cq values followed by stabilization over time. The colored lines are the piecewise regression lines for each miRNA, and the dots represent the means of the Cq values; each sample was tested in triplicate. MiRNA Cq values decreased initially and then stabilized over time (Suppl. Table 1).