A new method for in vitro RNA radioiodination with the aid of chloramine T has been developed. The 1251-labeled RNA preparations obtained by this method were uniformly labeled, nondegraded, and of high specific activity. The method is simple and the results are reproduced easily.
Several methods using radioactive isotopes have been developed for obtaining uniformly labeled RNA preparations with high specific activities. The best results were obtained when nucleic acids were radioiodinated (1). Currently, the most widely used method is that of Commerford (2) utilizing TlCL. Such labeling, however, takes place under stringent conditions. Therefore, an attempt was made to develop a new method for the preparation of a highly active, radioiodinated RNA under milder conditions. This was the basis for using chloramine T in our method that is known to be used for protein iodination.
Methods
Isolation of RNA. Liver RNA preparations were isolated from albino rats (3) from the Rappolovo Breeding Farm, Academy of Medical Sciences, Leningrad District, U.S.S.R. A260/A23,, and A260/A28,, ratios for all preparations used were between 2.0 and 2.2. The amount of RNA in samples was measured spectrophotometrically taking into account that the molar extinction coefficient, Ezso (p), is equal to 7800 (4).
CsCl gradient isopycnic centrifugation. RNA preparations were centrifuged on a stepwise CsCl gradient (4.0 ml each of concentrations of 48, 58, and 64%) containing 0.15 M NaCl and 0.1 M sodium acetate at pH 5.1. The samples were centrifuged at 35,000 rpm for 14 hr at 16ยฐC in a Beckman-Spinco 50L ultracentrifuge (U.S.A.) with a Ti 50 rotor. After centrifugation, 0. l-ml gradient fractions were collected from the tube bottoms. CsCl density ( pz5q in these fractions was calculated according to an equar Laboratory of Biochemistry.