Visualizing Nuclear mRNA by FISH

poly(A)RNA FISH was performed as described earlier^{66 (link)}. In brief, cells were treated with 1 mM auxin for 8 h, washed with PBS, fixed for 15 min in 4% PFA at RT, incubated in 70% ethanol for 16 h at 4 °C, washed with PBS, and incubated with wash buffer for 5 min at RT. Cells were then incubated with oligo(dT)-Quasar 670 probes (LGC Biosearch Technologies) for 4 h at 37 °C, followed by incubation with wash buffer at 37 °C for 30 min and then washed with PBS. Cells were then stained with 1.5 μg/ml Hoechst 33258 (Molecular Probes/Life Technologies) for 10 min, washed with PBS, and finally mounted in ProLong Gold antifade reagent (Life Technologies).
Individual RNAs were detected by FISH using ViewRNA Cell Plus (Thermo Fisher Scientific, Cat#88-19000-99) and corresponding RNA probes (Thermo Fisher Scientific, Cat# VX-01), according to the manufacturer’s instructions. Briefly, cells were seeded on 24-well plates with coverslips, processed once they reach 80% confluency, and fixed according to the ViewRNA Cell Plus protocol. Mab414 antibodies were used to detect NPC.
Cytoplasmic-to-nucleus (Cyt/Nu) ratio of mRNA foci was plotted with scatter plot function in Prism software. Data are expressed as mean value with standard deviations as error bars. Unpaired two-tailed Student’s t test has been applied for comparison of untreated and auxin-treated cells.