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/A260. 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.
Highlights
One of the most popular methods to measure the concentration of nucleic acids is ultraviolet (UV) absorbance spectroscopy
Ultraviolet spectroscopy is commonly used to quickly quantify nucleic acids in biology, but its use requires that an accurate extinction coefficient is known
Our methodology allows for mixtures of DNA and RNA to be digested simultaeously and provides separation of the ribonucleosides from the deoxyribonucleosides
Summary
One of the most popular methods to measure the concentration of nucleic acids is ultraviolet (UV) absorbance spectroscopy. With the development of nano spectrophotometers, as little at 2 mL of sample is adequate to determine the concentration and as little as 3 ng mLÀ1 can be detected within a matter of seconds.. The concentration of nucleic acids is calculated using the Beer– Lambert law (A 1⁄4 3 Â C Â l), where A is absorbance, determined by the spectrophotometer, 3 is the molar extinction coefficient of the measured sample, C is the concentration of nucleic acids in solution and l is the pathlength of the vessel in the spectrophotometer.. While Beer–Lambert's law is straightforward, there are other factors that can complicate the analysis of nucleic acids, including conformational changes in the structure, base-stacking, and base pairing These can cause a shi from the expected molar extinction coefficient relative to the simple sum of absorbance of individual nucleotides. This effect is referred to as either hypochromicity, which is a decrease in absorbance, or hyperchromicity, which is an increase in absorbance
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
