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This procedure is a modification of the original method published by Chirgwin et al. (1). The following protocol is especially adapted for small amounts of tissue and cells (1020mg wet weight) and results in undegraded RNA preparations, lg tissue or cells are homogenized with 8ml lysis buffer (4M guanidinium thiocyanate, 2% 2-mercaptoethanol, 0.1M Hepes pH 7.5, 0.5% (w/v) sarkosyl) using a motor driven teflon pestle (510 strokes, 1000 rpm) for cells or a dounce homogenizer (about 10 strokes) for tissue. The genomic DNA is sheared mechanically until the solution has lost its high viscosity. The homogenate is centrifuged at 10,000g for lOmin at 10-15°C. RNA is precipitated from the supernatant by slowly adding 0.025 vol IN acetic acid and 0.5 vol ethanol while vortexing the sample, and then put on ice for several hours or overnight. RNA, denatured proteins, and contaminating DNA are then sedimented by centrifugation at 10,000g for 20 min at 4°C. The sediment is dissolved in 3ml 0.1M Hepes pH 7.5, 25 mM EDTA, 1% SDS) and heated to 60°C. After centrifugation (lOmin, 10,000g), the aqueous phase is extracted by repeated phenol/chloroform extractions at 60°C (10 min incubation) until no interphase is visible. Reextraction of the interphase is recommended. The RNA is finally collected by standard ethanol precipitation. The volume of the sediment is estimated and dissolved in 4-5 vol 100 mM Hepes pH 7.5. The same volume of 5M LiCl is added dropwise while vortexing (avoiding the immediate precipitation of nucleic acids), and the sample is put on ice for at least 4 hrs or overnight. The RNA is collected by centrifugation at 10,000g for 20 min. Reprecipitation with lithium chloride further reduces the amount of contaminating DNA. The RNA is finally washed in ice cold 75% ethanol to reduce the lithium salt, which inhibits glyoxylation (2). To obtain an optimal A : A ratio, a proteinase K step can be performed as described by Maniatis et al. (3). To remove residual traces of DNA, inclusion of DNase I (166ug/ml) in this step according to Tullis and Rubin (,3) is advisible.

A universal and efficient protocol for the isolation of RNA from tissues and cultured cells