Determination of RNA degradation by capillary electrophoresis with cyan light-emitted diode-induced fluorescence

Abstract

RNA integrity plays an important role in RNA studies because poor RNA quality may have a great impact on downstream methodologies. This study proposes a cost-effective, rapid, and sensitive method for determining RNA integrity based on capillary electrophoresis that utilizes a cyan light-emitted diode-induced fluorescence as a separation tool. The capillary was initially coated with 0.1% Poly(vinylpyrrolidone) (Mave 1,300,000Da) to reduce electroosmotic flow and avoid RNA adsorption. When the capillary was filled with 0.4% poly(ethylene) oxide (Mave 4,000,000) and a nucleic acid-specific fluorescent dye, SYTO 9, the baseline separation of the 18S and 28S ribosomal RNAs (rRNAs) in total RNA was accomplished within 15min. The lowest detectable concentration for the 18S and 28S rRNAs was estimated to be 50pg/μL. Some peaks longer than the 28S rRNA that migrated slowly were observed as long as the initial total RNA concentration was optimized. The temperature-induced degradation of the large RNA fragments (longer than the 28S rRNA) was faster than that of 18S rRNA and 28S rRNA. These large RNA fragments may serve as a promising marker for testing RNA integrity compared to the traditional method.