Nuclear mRNA Export Measurement Using FISH in iPSC-Derived Neurons

Nucleocytoplasmic transport (NCT) across the nuclear envelope plays a critical role in maintaining cellular homeostasis. Impaired NCT has been shown to be implicated in aging and many neurodegenerative diseases, including dystonia, amyotrophic lateral sclerosis, Alzheimer’s disease, and Huntington’s disease. To determine the pathogenesis underlying the impaired NCT-related diseases, it is critical to develop reliable approaches to measure the cellular NCT activities, including nuclear mRNA export. In this chapter, we describe a protocol to measure the nuclear mRNA export by using fluorescent in situ hybridization (FISH). Digoxigenin -labeled oligo-dT probes are used to detect poly(A) RNAs (mRNA). FISH combines with immunostaining of specific markers, such as microtubule-associated protein 2 to identify the soma of cultured neurons, and the DNA dye Hoechst 33342 to define the nucleus of target cells. The nuclear mRNA export activities could be measured by the subcellular distribution of mRNAs at a single-cell level in different cultured cells and tissues.