1. ONE STEP-RNA Reagent is a ready-to-use reagent for the isolation of total RNA from cells and tissues. The reagent, a mono-phasic solution of phenol and guanidine isothiocyanate, is an improvement to the single-step RNA isolation method developed by Chomczynski and Sacchi. During sample homogenization or lysis, ONE STEP-RNA Reagent maintains the integrity of the RNA, while disrupting cells and dissolving cell components. Addition of chloroform followed by centrifugation, separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase. After transfer of the aqueous phase, the RNA is recovered by precipitation with isopropyl alcohol. After removal of the aqueous phase, the DNA and proteins in the sample can be recovered by sequential precipitation. Precipitation with ethanol yields DNA from the interphase, and an additional precipitation with isopropyl alcohol yields proteins from the organic phase. Co-purification of the DNA may be useful for normalizing RNA yields from sample to sample.
2. This technique performs well with small quantities of tissue (50~100 mg) and cells (5×106), and large quantities of tissue (³1 g) and cells (>107), of human, animal, plant, or bacterial origin. The simplicity of the ONE STEP-RNA Reagent method
allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA isolated by ONE STEP-RNA Reagent is free of protein and DNA contamination. It can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, and molecular cloning. For use in the polymerase chain reaction (PCR), treatment of the isolated RNA with amplification grade DNase I is recommended when the two primers lie within a single exon.
3. ONE STEP-RNA Reagent facilitates isolation of avariety of RNA species of large or small molecular size. For example, RNA isolated from rat liver, electrophoresed on an agarose gel, and stained with ethidium bromide, shows discrete bands of high molecular weight RNA between 7 kb and 15 kb in size, (composed of mRNA’s and hnRNA’s) two predominant ribosomal RNA bands at ~5 kb (28S) and at ~2 kb (18S), and low molecular weight RNA between 0.1 and 0.3 kb (tRNA, 5S). The isolated RNA has an A260/280 ratio of 1.6~1.8 when diluted into distilled water. The expected yield of RNA per mg of tissue is: liver and spleen, 6~10mg; kidney, 3~4mg; skeletal muscles and brain, 1~5mg; placenta, 1~4mg. The expected yield of RNA from 1×10 6 Cultured cells is: epithelial cells, 8~15mg; fibroblasts, 5~7×mg.