Comparison of Three Magnetic Bead Surface Functionalities for RNA Extraction and Detection

Abstract

Magnetic beads are convenient for extracting nucleic acid biomarkers from biological samples prior to molecular detection. These beads are available with a variety of surface functionalities designed to capture particular subsets of RNA. We hypothesized that bead surface functionality affects binding kinetics, processing simplicity, and compatibility with molecular detection strategies. In this report, three magnetic bead surface chemistries designed to bind nucleic acids, silica, oligo (dT), and a specific oligonucleotide sequence were evaluated. Commercially available silica-coated and oligo (dT) beads, as well as beads functionalized with oligonucleotides complementary to respiratory syncytial virus (RSV) nucleocapsid gene, respectively recovered ∼75, ∼71, and ∼7% target RSV mRNA after a 1 min of incubation time in a surrogate patient sample spiked with the target. RSV-specific beads required much longer incubation times to recover amounts of the target comparable to the other beads (∼77% at 180 min). As expected, silica-coated beads extracted total RNA, oligo (dT) beads selectively extracted total mRNA, and RSV-specific beads selectively extracted RSV N gene mRNA. The choice of bead functionality is generally dependent on the target detection strategy. The silica-coated beads are most suitable for applications that require nucleic acids other than mRNA, especially with detection strategies that are tolerant of a high concentration of nontarget background nucleic acids, such as RT-PCR. On the other hand, oligo (dT) beads are best-suited for mRNA targets, as they bind biomarkers rapidly, have relatively high recovery, and enable detection strategies to be performed directly on the bead surface. Sequence-specific beads may be best for applications that are not tolerant of a high concentration of nontarget nucleic acids that require short RNA sequences without poly(A) tails, such as microRNAs, or that perform RNA detection directly on the bead surface.

Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA

“…In addition, it can be readily applied to study RNA metabolism, such as RNA capping, RNA decapping and RNA decay. Notably, when combined with transcript-specific purification technology (76), it enables quantification of cap structures in specific transcripts and thus studies of transcript-specific capping and decapping, and gene-specific regulation. Finally, it permits investigations into the roles that cap-binding proteins, such as eIF4E and CBC, may play in the control of gene expression (77).…”

Combined ultra-low input mRNA and whole-genome sequencing of human embryonic stem cells

“…It has been shown that chemical conditions can also be changed to preserve proteins in their native state for further proteomic analyses [ 37 ]. However, the abovementioned 3′ skew bias has been faced in different approaches, like the cDNA transcription using SMARTer [ 28 ] or the additional mRNA enrichment by its 5′cap [ 38 , 39 ]. The latter procedure also deals with the intricate capturing of mRNAs with short poly-A tails since its length might be influenced in the course of translational control [ 40 ].…”

Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics

“…The first magnetic particle used for extraction consisted of an iron-oxide core covered by functional carboxylic group, which then binds DNA or RNA [81]. Since then, many modifications have been made using different surface functional group, such as sulphate, amino, and hydroxyl groups [82]. Besides these functional groups, preactivated magnetic beads with different functional groups are available such as tosyl or epoxy groups [78, 83].…”