Protocol to validate custom-designed BaseScope probes using cell-free synthesized protein lysates and in vitro-transcribed purified mRNA.

BaseScope is an ultra-sensitive in situ hybridization technique specifically designed for detection of rare, short RNA molecules, particularly enabling co-detection of exon junctions in splice variants. Its use is cost intensive due to required protocol optimization for each tissue, especially when target expression is very low. Here, we present a protocol to validate functionality of custom-designed BaseScope probes by utilizing cell-free synthesized protein lysates and in vitro-transcribed purified mRNA as positive controls. We detail steps for slide preparation, probe hybridization, signal amplification, and detection.