One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST)

Abstract

The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes “pinned” aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier, termed Immiscible Filtration Assisted by Surface Tension (IFAST). Nucleic acids in biological samples are bound to paramagnetic particles and then drawn across the IFAST device (or array of IFAST devices) using a magnet. While the strength of the IFAST barrier is suitable for separation of nucleic acids from lysate in its current embodiment, its permeability can be selectively adapted by adjusting the surface tensions/energies associated with the cell lysate, the immiscible phase, and the device surface, enabling future expansion to other non-nucleic acid applications. Importantly, processing time is reduced from 15–45 minutes to less than 5 minutes while maintaining purity, yield, and scalability equal to or better than prevailing methods. Operation is extremely simple and no additional lab infrastructure is required. The IFAST technology thus significantly enhances researchers’ abilities to isolate and analyze nucleic acids, a process which is critical and ubiquitous in an extensive array of scientific fields.

Both LRP5 and LRP6 Receptors Are Required to Respond to Physiological Wnt Ligands in Mammary Epithelial Cells and Fibroblasts

“…IP complexes were purified using the IFAST method (illustrated in Fig. 7A) (36). Immune complexes were eluted in the output well in PBS containing 0.01% Tween 20 (17 l).…”

“…We suspected it could be better at capturing low affinity and short lived complex components. This technique also dramatically reduces the purification time (36). 4 A scheme is shown in Fig.…”

Design criteria for developing low-resource magnetic bead assays using surface tension valves

“…In a recent report of a 96-well plate mRNA extraction assay analogous to our continuous tubing design, Berry et al use low concentrations of Triton X-100 to reduce the interfacial tension at the solution/valve interface. 13 The group reported that the addition of 0.01-0.1% Triton X-100 did not interfere with the mRNA binding chemistry yet facilitated the magnetic transfer of the beads across the immiscible phase.…”

“…This inverted solid phase format based on magnetic beads and stationary fluids separated by surface tension forces has been developed for a number of applications. [11][12][13][14][15] Other laboratory-based magnetic bead assays, such as enzyme-linked immunosorbent assays (ELISAs) or onbead isothermal polymerase chain reaction (PCR), could potentially benefit from the simplicity and flexibility of this format for applications in low-FIG. 1.…”

DNA/RNA Preparation for Molecular Detection

“…A disadvantage is that it takes about 4 h to run. A similar idea not currently in a product is that NA-bound beads could be moved through an immiscible liquid wash phase to remove contaminants (70 ).…”