Optimization of the determination of RNA nucleotide composition by ultraviolet spectral analysis

Abstract

We have optimized the spectrophotometric method for the quantitative analysis of a tetranucleotide mixture (for example an RNA hydrolysate) by carefully choosing the wavelengths and the pH at which the absorbance measurements were made, and estimated the error made on the calculated concentration of each nucleotide. The most convenient method involved the measurement of only four absorbances (at 228 nm at pH 13; 200, 212 and 284 nm at pH 2). If the sensitivity of the spectrophotometer is 0.001 unit absorbance, the absolute error made on the calculated percentage of each nucleotide is lower than 0.33%.