FIGURE 5:
The separation of nuclear and cytoplasmic RNA. (A) The fidelity of nuclear and cytoplasmic RNA separation was assessed by qRT-PCR quantification of RNAs known to be restricted to the nucleus or to the cytoplasm. The small nuclear RNA 7SK was the control for the nuclear fraction. Cytochrome B mRNA is a mitochondrially encoded transcript and not present in the nucleus, making it a good cytoplasmic RNA marker. Ct values were normalized using miniUAA1 mRNA from yeast added as an internal standard to each fraction after the separation of nuclei and cytoplasm. There was very little nuclear contamination of cytoplasmic RNA and very little cytoplasmic contamination of nuclear RNA. Treatment of cells with 20 μM PI3 kinase inhibitor LY294002, 5 μM AKT inhibitor VIII, or 100 nM mTOR inhibitor rapamycin did not affect the partitioning of either 7SK RNA or cytochrome B mRNA. Each bar represents the average of two independent experiments in which triplicates were analyzed. Error bars represent the SE of the mean. ns, not significant. (B) The quality of the nuclear–cytoplasmic fractionation was also assessed by Western blotting using the large subunit of RNA polymerase II as a nuclear control and γ-tubulin as a cytoplasmic control. The results confirmed that nuclear and cytoplasmic material was cleanly separated.