Extraction of Cellular and Tissue RNA

Total undegraded cellular RNA, largely free of contaminating DNA, can be rapidly and easily isolated from homogenized tissue or cultured cells using acid pH-guanidinium thiocyanate/phenol/chloroform extraction (1 ). Commercially available acid phenol/guanidinium thiocyanate solutions (2 ), such as RNAzol™ B (Cinna Scientific, Cleveland, OH) and TRIzol (Gibco-BRL, Gaithersburg, MD), allow one-step, complete solubilization of tissue or cells under conditions that inhibit RNAse activity. By subsequent addition of chloroform, phase separation is forced to occur and RNA is extracted into an aqueous phase, separated from lower organic phase by an insoluble protein interphase. Guanidinium thiocyanate remains in the aqueous phase and so continues to act as an RNAse inhibitor by disrupting protein-nucleic acid interactions. However, the protein interphase is still rich in RNAse and, on accidental recovery with the aqueous phase, may result in degradation of the RNA. To eliminate this contamination, aqueous phase can be re-extracted in fresh phenol/chloroform/isoamyl alcohol solution (see Note 1 ) such as PCI reagent (5 Prime→3 Prime [Boulder, CO]) and spun through a barrier material that separates phases of greater and lesser density. The lighter aqueous phase does not penetrate the barrier, whereas the organic phase and any residual protein interphase migrate through the barrier to become trapped underneath. Heavy grade phase-lock gel tubes (5 Prime→3 Prime) are suitable for procedures using guanidinium thiocyanate, which imparts higher density to the aqueous phase (3 ).