A method for quantitative spatial gene expression analysis of mammary gland tissue morphological changes associated...

RNAscope is a novel quantitative in situ gene expression assay that allows single molecule visualization and preservation of spatiotemporal and cell specific information across intact tissue samples. This method has utility to measure gene expression using intact tissue; thus, allowing quantification in a spatial and cell type specific manner, which can be valuable to complement single cell sequencing approaches that use disaggregated tissue. The accurate quantitative capacity of RNAscope depends on retention of RNA integrity which can be lost with extensive or poor tissue handling procedures. This protocol outlines a method of processing and carmine staining mammary gland whole mounts, which is commonly used to identify developmental and reproductive morphologies or disease lesions, followed by excision of tissue areas of interest and RNAscope on tissue sections. This method preserves both RNA integrity and accurate quantification of the RNAscope technology, while bypassing a need for labor-intensive screening of serial sections to identify unpalpable disease lesions induced by environmental exposures at various stages of reproductive development.