Sucrose and cesium chloride gradient centrifugation are extremely widely used techniques for the fractionation of RNA and ribonucleoprotein particles. A variety of methods have been used to separate high molecular-weight material from gradient fractions as a preliminary to scintillation counting (l-3), but in the absence of large amounts of labeled acid-soluble material direct counting of samples using an aqueous scintillation mixture is the most convenient technique. The most popular systems have been those containing dioxane and napthalene with PPO and POPOP as primary and secondary scintillants (4-7). These mixtures are, however, expensive, the counting efficiency drops with increasing water concentration and it has been reported that the efficiency of counting RNA (but not of nucleotides) is impaired by the presence of sucrose (8). Recent surveys have shown that the scintillant butyl-PBD (2-(4'-t-butylphenyl) -5-(4"-biphenylyl) -1,3,4-oxadiazole) is outstanding, especially in its resistance to quenching (9,lO). It has also been found that the detergent Triton X-100 wilI form stable emulsions with toluene and water in which scintillation counting can be carried out with high efficiency (11). In this communication we report investigations on the counting of RNA and ribonucleoprotein particles in the presence of density-gradient solutes in a scintillation mixture containing butyl-PBD and Triton X-100.
The scintillation solution was prepared by dissolving 31.5 g of butyl-PBD (Koch-Light Ltd., Colnbrooke, Bucks.) in a mixture of 3 liters of toluene, 0.5 liter of methanol, and 1.5 liter of Triton X-100. The three latter chemicals were obtained from BDH Ltd. (Poole, Dorset) and were of AnalaR grade. Polysomes were prepared from rats injected 4 hr previously with [6-W] -or [5-3H ] erotic acid (Radiochemical Centre, Amersham) by the magnesium-precipitation method of Leitin and Lerman (12) and RNA extracted with sodium dodecylsulfate and 270