Complete enzymatic digestion of double-stranded RNA to nucleosides enables accurate quantification of dsRNA

Abstract

The rapid growth of research focusing on RNA, especially for RNA interference applications, has created a need for a robust method that can accurately determine the concentration of long dsRNA. As it is difficult to find a source for pure dsRNA reference material, the most common method for quantitation is using a reversed-phase HPLC method to determine purity, which is linked to a calibration curve prepared by measurements obtained using UV absorbance at 260 nm. In this study we developed a nucleic acid digestion method that can digest both double- and single-stranded RNA and DNA to nucleosides. A reversed-phase HPLC/UV method was used to separate and quantitate the monomeric nucleosides. Using this method, we were able to calculate the absorptivity coefficient (proxy for the extinction coefficient) for dsRNA to be 45.9 ± 0.52 μg mL⁻¹/A_260. This value agrees with the one report we were able to find but uses an orthogonal method. Moreover, this study allowed us to understand that sequence design can dramatically change the extinction coefficient of the molecule. For molecules with ssRNA overhangs, we observed a 5% reduction in the calculated extinction coefficient.