Trizol Tm : a New Reagent for Optimal Single - Step Isolation of Rna

Piotr Chomczynski Department of Internal Medicine School of Medicine University of Cincinnati Cincinnati, Ohio 45267 Progress in many fields of basic and clinical research depends on the availability of high-quality RNA. Of the many methods for isolating RNA (reviewed in 1 and 2), a single-step RNA isolation method based on guanidine isothiocyanate/phenol/chloroform extraction (3) has been growing in popularity because it dramatically reduces the amount of time required to isolate RNA without sacrificing its quality. TRIZOLTM Reagent is an improved version of the single-step RNA isolation method. TRIZOL Reagent (patent pending) is a monophasic solution of phenol and guanidine isothiocyanate. During sample homogenization or lysis, TRIZOL Reagent maintains the integrity of the RNA, while disrupting cells and solubilizing cell components. Addition of chloroform, followed by centrifugation, separates the solution into an aqueous phase and an organic phase. The RNA remains in the aqueous phase and is recovered by precipitation with isopropanol. The DNA and proteins remain in the organic phase and can be recovered by sequential precipitation (4). Precipitation with ethanol yields DNA from the interphase, and an additional precipitation with isopropanol yields proteins from the organic phase (4). This technique can be used with small quantities of tissue (50 to 100 mg) and cells (5 × 106) and large quantities of tissue (≥1 g) and cells (>107), of human, animal, plant, or bacterial origin. The simplicity of the TRIZOL Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. The RNA can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, molecular cloning, and polymerase chain reaction (PCR).