Quantitative ultrasensitive bright-field RNA in situ hybridization with RNAscope.

RNA in situ hybridization (ISH) can provide valuable morphological context for molecular markers on one hand and enable morphological analysis in molecular context on the other hand. It has become increasingly important, thanks to increasing interest in new biomarkers and noncoding RNAs in both research and clinical applications. We have developed an ultrasensitive RNA ISH technology, RNAscope, employing a unique probe design strategy that allows target-specific signal amplification while suppressing background noise. This approach enables single RNA molecule detection in formalin-fixed paraffin-embedded (FFPE) specimens under standard bright-field microscopy and is capable of multiplex detection at the single cell level. After staining, target-specific signals appear as punctate dots present in individual cells in well-preserved tissue morphological context, which facilitates both semiquantitative manual scoring and software-assisted quantitative analysis. Here, we present detailed protocols of RNAscope for FFPE tissue sections. The step-by-step protocols describe tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis. We also highlight the critical steps for ensuring successful staining.