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.

© The Royal Society of Chemistry 2011

Figures

Fig. 1

Comparison of the protocols for conventional PMP-based NA purification and IFAST device. The IFAST method reduces the number of processing steps from 18 to 6 (67%) and the total purification time from 17.7 minutes to 4.1 minutes (77%), largely through the elimination of repetitive washing steps. More details on each step are given in Fig. S1†.

Fig. 2

IFAST device design and operation. Large arrays of devices, such as the 114 device array shown in (A), can be operated in parallel using arrays of magnetic strips. The IFAST platform consists of three wells connected in series by microchannels. The central well is loaded with an immiscible oil phase to separate cell lysate containing NA-binding magnetic particles from nuclease-free elution buffer (B). A magnet is utilized to draw NA-bound particles through the immiscible phase (C) and into elution buffer (D). The total time to operate actual device is only 10 seconds (E). Increasing the concentration of a detergent (Triton X-100) reduces the interfacial energy with the oil phase (Chill-Out Liquid Wax), promoting carryover of liquid across the immiscible phase barrier (F). The various regimes of PMP transfer are defined by the red dashed lines and photographs of representative traverses are given in the graph, with an interfacial energy of −3 to 15 mN m−1 resulting in ideal transfer of PMPs. By reducing the surface energy between the oil and the device surface, the ideal regime can be expanded, as defined by the blue dashed lines, to encompass the entire tested range of detergent concentrations.

Fig. 3

Optimization of the immiscible phase interface. Using olive oil as the immiscible phase results in a favorable outcome relative to liquid wax despite its lower interfacial energy with a detergent-rich lysis buffer (A). The difference in contact angle between lysis buffer and glass submerged in both oils elucidates differences in interfacial energies. Data encompass 6 measurements for each oil and error bars represent standard deviations (B). Side view of PMP aggregate traverse is shown both schematically (C) and photographically (D) to illustrate the selective extension of lysate between the oil and the floor of the device.

Fig. 4

IFAST performance. Efficiency was calculated by amplifying multiple genes (P0 and ERα) from a serial dilution of IFAST-purified NA and fitting the given equation to the data (A). The yield was determined by amplifying three genes (P0, ERα, and GR) from mRNA from ~56 000 breast cancer epithelial cells (MCF-7) using both the IFAST and commercial kits to isolate the NA. Data represent three independent experiments performed in duplicate and error bars represent standard deviation (B). The implementation of PMPs coated with oligo-dT nucleotides selectively captured mRNA (as quantified by amplifying the P0 gene) from cell lysate (C). Sensitivity was measured by purifying a series of cell concentration via IFAST and amplifying multiple genes (P0 and ERα). Data represent three experiments for each cell concentration and error bars represent standard deviations of the data (D).