Refining A Protocol for Preparation for RNAScope of Swine and Pediatric Biopsy Samples

Abstract

Bone and cartilage samples can be difficult to prepare for histological, immunohistochemical, and molecular analysis techniques due to their cellular characteristics, the need for decalcification, and issues with tissue adherence to slides. Our objective was to develop a protocol for RNAscope assessment of formalin-fixed paraffin-embedded (FFPE) bone and cartilage samples that minimizes tissue loss and preserves RNA adequately. We evaluated multiple protocols to optimize the presence of high quality RNA based on the relative expression of medium and low expression markers by RNAScope assay. Through an iterative process, we determined that our protocol 1) was effective based on the evaluation of positive controls, 2) that pre-baking slides at 34°C for 6 improved tissue adherence, and 3) that RNA was present in adequate amounts within human osteochondral tissue to be detected using our protocol. Collectively, these findings will guide future protocol optimization for RNAScope evaluation of RNA in FFPE bone and cartilage samples by our laboratory.