Since the report by Richards et al. ( 1) , the analysis of ribonucleic acids (RNA) on polyacrylamide gels has become widely used because of the possibility of an exceedingly sharp and perfect fractionation from a mixture. The methods used for detection of RNA after fractionation showed a various degree of sensitivity and stability. The majority of methods of RNA detection by staining are based on the ability of RNA to form complexes with certain dyes. Of the latter, acridine orange (1)) methylene blue (2), pyronine Y (also designated as pyronine G) (3,4), toluidine blue 0 (5)) or 'Stains-all" (6) were either used for staining the gels after electrophoresis or added to the RNA sample before electrophoresis. A rapid and excellent method is direct densitometric evaluation of the gels by scanning in ultraviolet light (7)) but the necessary equipment is not available everywhere; it may be avoided, provided a suitable staining method is available.
Several of the hitherto used methods of RNA staining were tested without satisfactory results (insufficient staining of RNA; heavily stained background after destaining; labile binding of the dye on RNA; or photosensitivity). In searching for optimal conditions, modifications of the individual methods were tested. A staining method, giving a stable and photoresistant staining of RNA, in addition to being highly sensitive, was elaborated. The results are presented below.
Disc electrophoresis was carried out according to Loening (7). A 4% gel, calculated only on acrylamide monomer, was employed. N,N'-Methylenebisacrylamide (BIS) was used in a 5% concentration with respect to acrylamide. Acrylamide and BIS were used after recrystallization from benzene and chloroform, respectively (7). Glass tubes 7 cm long of 0.5 cm i.d. were employed; the height of gel columns was 5.0 cm. The test sample was a mixture of RNA's obtained by phenol extraction from Krebs II ascites carcinoma mouse cells, using a slight modification of the method of Gierer and Schramm (8). In sucrose gradients, these RNA's 304