Optimization of microRNA extraction from the plasma of the common carp.

Abstract

Efficient and safe extraction of microRNAs (miRNAs) from biological samples is pivotal for genetic regulation studies and biotechnological applications. This study focuses on optimizing the microRNA extraction process from the plasma of common carp, a significant species in aquaculture. Recognizing the limitations and hazards of commercial extraction kits, which often employ toxic chemicals like phenol and chloroform, we sought to develop a safer and more effective alternative. Our optimized protocol utilizes guanidinium isothiocyanate (GITC) and sarkosyl, omitting hazardous substances. We explored several parameters including GITC concentration, the addition of sarkosyl, and the role of sodium chloride in enhancing miRNA yield. Our findings demonstrate that optimal conditions involve a GITC concentration of 4.2 M, a 3% sarkosyl concentration, and the use of sodium chloride at 0.5 M. We also investigated the utility of glycogen as a nucleic acid carrier, finding 160 µg to be the optimal concentration. Comparative analysis with commercial kits indicated our method provides higher miRNA yields with reduced cycle threshold values, underscoring the effectiveness of our custom protocol. This optimized approach not only enhances miRNA recovery but also emphasizes safety and cost-effectiveness, making it a valuable method for both research and practical applications in aquaculture.