NUCLEOTIDE COMPOSITION OF RIBONUCLEIC ACID BY SPECTRAL ANALYSIS AND THIN-LAYER CHROMATOGRAPHY

Improved spectrophotometric and thin-layer chromatographic methods for the rapid determination of RNA nucleotide composition are described. These methods are suitable for quantitative analysis of as little as 120 μg of unlabeled RNA or smaller amounts of isotopically labeled RNA. Both procedures employ a cation-exchange resin, Dowex 50, for the separation of alkaline hydrolyzed RNA into two nucleotide pairs of 3′(2′)-ribonucleoside monophosphates: UMP-GMP and AMP-CMP. This separation is achieved within 30-45 min. With the spectrophotometric method, the nucleotides in each pair are accurately determined by utilising the differences in absorbance of the nucleotide pairs at selected wavelengths. With the alternate thin-layer chromatographic method, complete separation of 32P-labeled nucleotides in each mixture is obtained on microcrystalline cellulose layers in 2.5 hr and the single nucleotides are accurately determined by radioactivity measurements. The nucleotide compositions of yeast RNA and 32P-labeled chick embryo fibroblast cell RNA were determined with a high degree of accuracy and were found to agree with previously reported values. © 1968.