Coring vs scrolls: Inclusion of the muscularis layer on tissue scrolls of the urinary bladder leads to inaccurate miRNA expression

Abstract

Formalin fixation with paraffin embedding (FFPE) is a method used to preserve tissues. FFPE tissues can be sampled through tissue cores or scrolls. In the study of urothelial carcinomas (UC), the use of a biopsy needle to acquire cores has the advantage of sampling the targeted superficial epithelial cell layer. In contrast, tissue scrolls are collected using a microtome, which includes surrounding tissues such as the muscularis layer. While both methods allow sample collection to extract small molecules, such as miRNAs, there is a lack of literature comparing these methods. MiRNAs are non‐coding RNAs responsible for gene regulation, altered in cancer. UC is the most common malignant bladder tumor in dogs, with 20,000 cases annually. The diagnosis of UC is challenging due to the small sample size of biopsies acquired by endoscopy. This project aimed to compare miRNA expression between the FFPE collection methods in UC (n=3) and healthy bladder (n=1) from dogs to understand the effect of including tissues surrounding a lesion in scrolls. MiRNA was extracted using a commercial kit, and the expression was achieved through RT‐qPCR. Cores targeting the epithelia and the muscularis layer were compared to their corresponding scrolls. MiR‐214, miR‐152, miR‐181a, and miR‐39 were differentially expressed when comparing cores with scrolls and the muscularis layer, while miR‐1842 and miR‐143 were not. Comparison between methods within individual samples also showed differential expression for all miRNAs. These results show that the inclusion of the muscularis layer results in misleading miRNA expression and verifies cores as a more accurate technique in miRNA extraction and expression, supporting further miRNA biomarker research.