Selective and Efficient RNA Analysis by Solid-Phase Microextraction

摘要

In this study, a solid-phase microextraction (SPME) method was developed for the purification of mRNA (mRNA) from complex biological samples using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for quantification. The chemical composition of the polymeric ionic liquid (PIL) and a polyacrylate (PA) SPME sorbent coating was optimized to enhance the extraction performance. Of the studied SPME sorbent coatings, the PIL containing carboxylic acid moieties in the monomer and halide-based anions extracted the highest amount of mRNA from aqueous solutions, whereas the native PA fiber showed the lowest extraction efficiency. On the basis of RT-qPCR data, electrostatic interactions and an ion-exchange mechanism between the negatively charged phosphate backbone of RNA and the PIL cation framework were the major driving forces for mRNA extraction. The optimized PIL-based SPME method purified a high quantity of mRNA from crude yeast cell lysate compared to a phenol/chloroform extraction method. The reusability and robustness of PIL-based SPME for RNA analysis represents a significant advantage over conventional silica-based solid-phase RNA extraction kits. The selectivity of the SPME method toward mRNA was enhanced by functionalizing the PA sorbent with oligo dT(20) using carbodiimide-based amide linker chemistry. The oligo dT(20)-modified PA sorbent coating demonstrated superior extraction performance than the native PA sorbent coating with quantification cycle (Cq) values 33.74 +/- 0.24 and 39, respectively. The modified PA sorbent extracted sufficient mRNA from total RNA at concentrations as low as 5 ng mu L-1 in aqueous solutions without the use of organic solvents and time-consuming multiple centrifugation steps that are required in traditional RNA extraction methods.