Magnetic ionic liquids as microRNA extraction solvents and additives for the exponential amplification reaction

Abstract

The detection of microRNAs (miRNAs) from highly complex matrices has become an area of immense interest as their characterization in biological samples has been utilized for disease diagnosis and body fluid identification. However, conventional northern blotting miRNA detection lacks the sensitivity required to detect circulating miRNAs. Additionally, polymerase chain reaction-based methods for miRNA detection require modified oligonucleotides that are difficult to design. Exponential amplification reaction (EXPAR) is an isothermal amplification method used for miRNA detection that is simple to design but suffers from non-specific amplification that masks low concentration miRNAs. Previous studies have shown that magnetic ionic liquids (MILs) are a promising alternative to traditional nucleic acid extraction methods capable of preconcentrating DNA from complex matrices. In this study, three hydrophobic magnetic ionic liquids (MILs) were investigated as EXPAR additives and miRNA extraction solvents. The addition of MIL to the EXPAR buffer decreased the background signal from non-specific amplification and increased the reaction rate. Reactions containing MIL could detect miRNA at concentration levels down to 10 aM. In comparison, reactions that did not contain MIL could not discriminate 10 fM lethal-7a (let-7a) standards from the no trigger control (NTC). All three MILs extracted miRNA from 2-fold diluted plasma, artificial urine, and artificial saliva with only a 1 min dispersion step. By integrating the miRNA-enriched MIL into the EXPAR buffer, the extraction and detection of femtomolar concentrations of miRNA required only 10 min. In contrast, conventional spin column kits require at least 20 min to isolate miRNA, indicating that a dispersive MIL-based extraction is ideal for high throughput analysis of miRNA.