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.

中文翻译：

---

将双链RNA完全酶消化成核苷，可准确定量dsRNA

专注于RNA的研究的迅速发展，特别是对于RNA干扰应用，对可准确确定长dsRNA浓度的稳健方法的需求不断增加。由于很难找到纯dsRNA参考材料的来源，因此最常见的定量方法是使用反相HPLC方法确定纯度，该方法与通过使用260 nm的紫外吸光度获得的测量结果绘制的校准曲线相关。在这项研究中，我们开发了一种核酸消化方法，可以将双链和单链RNA和DNA消化成核苷。反相HPLC / UV方法用于分离和定量单体核苷。使用这种方法，我们能够计算出dsRNA的吸收系数（代表消光系数）为45.9±0.52μgmL^-1 /一₂₆₀这个数值与我们能够找到一个报告同意，但使用正交方法。此外，这项研究使我们理解序列设计可以显着改变分子的消光系数。对于具有ssRNA突出端的分子，我们观察到了计算的消光系数降低了5％。