RNA expression analysis on formalin-fixed paraffin-embedded tissues in TMA format by RNA in situ hybridization

The technique of RNA in situ hybridization, i.e., the detection of specific messenger RNA sequences within structurally intact cells or tissues is not widely used in biomedical research, because it can be cumbersome and technically challenging. However, it has a major advantage that warrants and sometimes even requires its application and the associated efforts. RNA in situ hybridization enables a detailed analysis of gene expression in the absence of a suitable antibody to the molecule encoded by the gene of interest. Within the wealth of RNA analysis technologies available nowadays, RNA in situ hybridization still is the only methodology that allows a precise localization of gene expression at a cellular level. This is particularly important if, e.g., new molecular markers or potential drug target molecules have to be analyzed in large cohorts of human tissues. In cancer research, it may be necessary to show that a newly characterized molecule is indeed expressed by the tumor cells themselves, rather than by any surrounding tissue. A protocol is presented here that has been routinely and successfully used on FFPE tissues assembled on a tissue micro array (TMA).