USE OF DIPHENYLAMINE AS A COLORIMETRIC REAGENT FOR RIBONUCLEIC ACID

RNA has been demonstrated to react with diphenylamine when acid hydrolysis is performed for 1 hour or more at 100°C. This reaction can be used for quantitative analysis of RNA, since there is a linear relationship between RNA concentration and absorbance. The reaction of RNA with diphenylamine can be quanlitatively distinguished from the reaction of DNA: the absorption spectrum of the RNA-diphenylamine reaction product has a maximum at 650 mμ, and a second, smaller peak at 490 mμ, while the DNA-diphenylamine reaction product has a single maximum at 605 mμ. It was found that, when mixtures of DNA and RNA are reacted with diphenylamine, the spectra reflect both the DNA:RNA ratios and the total amounts of nucleic acids. When the two-wavelength method of spectrum analysis was applied to such spectra, good agreement was found between actual and calculated values of nucleic acid concentrations. In this way, diphenylamine can be used for the simultaneous determination of the concentrations of DNA and RNA in mixtures. As is the case for the reaction of DNA with diphenylamine, it was found that the reaction of RNA is not altered by the presence of protein and that it involves primarily the purine nucleotides. The reaction of RNA with diphenylamine is discussed in relation to its possible analytical applications. © 1969.