Isolation of RNA from tissue
This protocol (CHOMCZYNSKI, 1993) allows the simultaneous recovery of RNA, DNA & protein from an aliquot of tissue or cells. This method involves lysis of cells with monophasic solution of guanidine isothiocynate and phenol. Addition of chloroform generates a second (organic) phase into which DNA & Proteins are extracted leaving RNA in aqueous supernatant. The RNA is precipitated from aqueous phase with isopropanol and can be further purified by chromatography on oligo dT- cellulose columns and/or used for northern blot hybridization, reverse transcription or RT-PCR. The yield of total RNA depends on the tissue or cell source, but it is generally 4-7 g/mg starting tissue or 5-10 g/ 10 6 cells. The DNA and Proteins can be isolated from the organic phase by sequential precipitation with ethanol and isopropanol respectively.