Determination of the Firefly Luciferase mRNA Concentration and Encapsulation Efficiency in Nanoparticles Using RiboGreen Assay

Abstract

Messenger RNA (mRNA) has been used as a powerful therapeutic in gene therapy and is used for infectious diseases. Novel delivery systems, such as lipid nanoparticles and reagents, are aimed at protecting mRNA from degradation. To quantify mRNA and to find its Encapsulation Efficiency, RiboGreen, a proprietary fluorescent RNA binding dye, was used for the detection and quantitation of RNA. The purpose of this study was to optimize a Ribogreen Assay for determination of Fluc mRNA Encapsulation Efficiency in nanoparticles, including Lipofectamine Messenger Max (LFMM) and solid Lipid Nanoparticles (LNPs). In the study, the effects of assay conditions associated with detergent (Triton) extraction of the mRNA were analyzed. We found that increasing the volume of LFMM to the weight of the mRNA ratio from 1.5 to 6 ul/ug resulted in the complete encapsulation of mRNA. Our initial 25 percent underestimate of free mRNA without LFMM (3.5 vs 4.7) was found to be due to poor replicates in the standard curve, and differing conditions in the sample vs standard (OptiMEM, Triton). By using identical conditions in the sample and standard and ensuring duplicate repeatability (5 percent), the 25 percent underestimate was reduced to 6 percent. The further influence of transfection reagent components needs to be completed. Future applications include finding the optimal setting (vector type, reagent components, and ratio) for the highest encapsulation efficiency of mRNA.