Single mRNA Detection Using MTRIPS-FISH

RNA localization and interactions within a cell can give valuable insight into the cell’s actions and reactions, especially during a disease state. Assays that give spatial information about RNA localization in cells are extremely useful as controls in interaction studies and also in providing functional understanding of cellular processes. For this purpose, Fluorescent In-Situ Hybridization (FISH) assays use fluorescently labeled strands of nucleic acids that are complementary to the RNA of interest to illuminate where the RNA is at that point in time. While much success has been achieved in visualizing single RNA molecules in live cells by the Santangelo Laboratory, FISH in fixed tissues with single-molecule specificity is more difficult to achieve. Here, we explore different methods of obtaining single-molecule specificity with previously validated MTRIPs molecules. We discovered that using prelabeled PNAs bound to the neutravidin protein lead to highly specific detection of single-molecule RNA, while allowing for other assays, such as Proximity Ligation, to occur simultaneously.Undergraduat