( gure 1). Analysis of ~5% of the RNA on ...)Efficient Cellular Fractionation for RNA ,,, and Protein Isolation

gure 1). Analysis of ~5% of the RNA on a 1.2% denaturing agarose gel showed a reproducible pattern of ribosomal RNA bands. While most of the mature 18S and 28S rRNAs were present in the total and cytoplasmic fraction, the precursor rRNAs (the two faint bands above the 28S rRNA band) were detected only in the total and nuclear fractions. Similar results were obtained for RNAs isolated from NIH-3T3, HeLa, and COS-7 cells (Figure 1).

Figure 1. Comparison of Total, Cytoplasmic, and Nuclear RNA and Protein From Different Cell Lines. RNA and protein were isolated from 1 x 10⁶ cells using the PARIS Kit. To allow direct comparison between total (T), cytoplasmic (C), and nuclear (N) fractions, the same proportion (~5%) of each prep was analyzed by denaturing agarose gel electrophoresis (NorthernMax-Gly Kit) or Western blot with anti-GAPDH antibody (Ambion). Nuclear vs cytoplasmic fractionation was performed in triplicate for 293 cells. M: Millennium Markers.

Efficient cellular fractionation at the protein level was confirmed by Western blot analysis with an antibody specific for the cytoplasmic GAPDH protein (Figure 1, bottom panels). Equivalent amounts of GAPDH protein were present in the total and cytoplasmic fractions with no detectable cross contamination in the nuclear fraction for the four cell types tested.

Isolation of Intronless Cytoplasmic mRNA
To further assess the usefulness of the PARIS Kit, we looked at the distribution of pre-mRNA and mRNA in total, cytoplasmic, and nuclear RNAs from HEK 293 cells. RNAs were treated with Ambion's

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