Amine-modified magnetic particles: An efficient tool for enhanced RNA extraction

Abstract

Magnetic particles are an effective tool for simple, time-saving, and labour-saving nucleic acid extraction. In this study, we investigated the isolation of nucleic acids (NA) using 11 variants of magnetic nanoparticles (MPs, 52 ± 6.8 nm) with a surface concentration of amine groups up to 20.8 nmol · mg−1. All results were compared with morphologically identical magnetic material modified with SiO2 grafted with (3-aminopropyl)triethoxysilane (APTES). The properties of these materials were characterized by transmission electron microscopy, scanning electron microscopy, dynamic and electrophoretic light scattering, and magnetometry. Concentrations of amine groups on MPs-APTES were determined by the chemical bind and release method with photometric quantification.The isolation potential of the proposed materials toward NAs was evaluated using gel electrophoresis with photometric determination of NAs concentrations and RT-qPCR. Our results show that the NAs yields of MPs-APTES are higher than the reference MPs-SiO2, regardless of the amine group concentrations. Although the total yield decreased with the concentration of amine groups, a different affinity towards genomic DNA (gDNA) was observed. A high concentration of grafted amine groups induced a preference for ribosomal RNA (rRNA) over gDNA and mediated effective NA elution. Densitometric image analysis of gDNA bands showed that NAs isolated by MPs-SiO2 contained significantly higher DNA levels than MPs with 1/32 %, 1/2 %, and 16 % APTES modification, which was subsequently confirmed by qPCR. Gene expression analysis performed by RT-qPCR revealed that unwanted gDNA contamination did not significantly affect the threshold cycles (Ct) of target genes when cDNA-specific primers were used, but may lead to overestimation when targeting genes with low expression and no possibility to design cDNA-unique primers. From a practical point of view, MPs-APTES provided better dose-dependent NA isolation performance with stable NA quality.