Meeting Abstract
In Situ Hybridization as a Localization Technique for miRNA in Austrofundulus limnaeus Cell Culture Kathleen K. Lukens, Josiah T. Wagner, Claire Riggs, Jason E. Podrabsky Department of Biology Center for Life in Extreme Environments Portland State University Portland, Oregon Understanding the role of microRNA (miRNA) expression and regulation is invaluable in modeling the cellular mechanisms associated with anoxia tolerance in the annual killifish Austrofundulus limnaeus. Development of an in situ hybridization (ISH) technique capable of specific and high resolution localization of target miRNA in cultured A. limnaeus cells would provide information about the location of miRNA in different sub-cellular compartments during periods of anoxia. ISH in the A. limnaeus cell culture system is a new technique and our objective is to develop a reliable protocol that can label with high specificity and localize within the structures of individual cells. Using immunohistochemistry (IHC) to fluorescently co-label the nuclei and mitochondria in conjunction with ISH can provide a map of miRNA transport and expression within the cell. Here, we use β-actin as positive control for RNA expression in order to optimize the ISH technique for A. limnaeus cell culture. Our protocol is suitable for both aerobic and anoxic cells, and accommodates IHC co-labeling of mitochondria and nuclei. Additionally, we demonstrate success in labeling for two expressed miRNAs in the same cell culture. Our results suggest differences in expression of target miRNA between anoxic and aerobic cells, thus indicating that microRNA expression and transport may play roles in the regulation of anoxia-induced quiescence in A. limnaeus embryos. Further development will be needed to improve specificity and reduce background labeling using this technique.
