Rapid Loss of RNA Detection by in Situ Hybridization in Stored Tissue Blocks and Preservation by Cold Storage of Unstained Slides

Objectives Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years. Methods To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic's RNAscope assay. Results We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases (<1 year old) at -20°C can preserve hybridization signals significantly better than storing the blocks at room temperature and cutting the slides fresh when needed. Conclusions We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.