Abstract
This protocol allows the simultaneous recovery of RNA, DNA, and protein from an aliquot of tissue or cells by lysis of cells with a monophasic solution of guanidine isothiocyanate and phenol. Addition of chloroform generates a second (organic) phase into which DNA and proteins are extracted, leaving RNA in the aqueous supernatant. The DNA and proteins can be isolated from the organic phase by sequential precipitation with ethanol and isopropanol, respectively. The DNA recovered from the organic phase is ∼20 kb in size and is a suitable template for PCRs. The proteins, however, remain denatured as a consequence of their exposure to guanidine and are used chiefly for immunoblotting. The RNA precipitated from the aqueous phase with isopropanol can be further purified by chromatography on oligo(dT)-cellulose columns and/or used for Northern blot hybridization, reverse transcription, or RT-PCRs. The yield of total RNA depends on the tissue or cell source, but it is generally 4-7 µg/mg starting tissue or 5-10 µg/106 cells. The A260/A280 ratio of the extracted RNA is generally 1.8-2.0.
Published Version
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