Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells

It often is desirable to "prefractionate" RNA before analysis. Ordinarily, this can only be done with tissue culture cells, although it is possible to isolate nuclei and cytoplasm from certain "soft" tissues such as liver and white blood cells. This protocol describes a method for separating nuclei from the cytoplasm that can be used for many tissue culture types. This procedure also is useful for cells grown in suspension or for adherent cells. The procedure relies on swelling in hypotonic buffer, subsequent gentle homogenization, and centrifugation. This method is not appropriate for material (e.g., bacteria, yeast) that has high intrinsic RNase activity, or tissues that are difficult to solubilize, such as muscle tissue or plant material.