To reduce the volume of solutions used, increase throughput, and decrease the amount of time to extract total RNA from cells, this paper shows the significance of using a microfluidic device in combination with magnetic bead-based solid phase extraction for the miniaturization and simplification of RNA purification. Here, we focus on total RNA extraction from white blood cells (WBCs), which requires isolation of white blood cells from whole blood, lysis of white blood cells, DNase treatment to degrade the DNA, and finally, purification of the RNA. Schein ( Totowa Humana Press Inc., 2001), pp. Pasloske, “ Ribonuclease inhibitors,” in Nuclease Methods and Protocols, 160th ed., edited by C. RNA isolation and purification, though, has further challenges as RNA molecules are less stable than DNA, and are degraded by ribonucleases (RNases) that are present on surfaces and in the air. Automated equipment, large numbers of personnel, sufficient quantities of reagents, equipment for cold storage, and more are required for high-quality nucleic acid extraction from multiple samples within a reasonable timeframe. limit the performance of nucleic acid preparations to well-resourced laboratories. Thatcher, “ DNA/RNA preparation for molecular detection,” Clin. When analyzing multiple peoples' biofluids for research studies or clinical diagnoses, the large number of preparation steps, the need for cold storage, and the lack of low-cost automation 1 1. The isolation and purification of nucleic acids from a biofluid such as blood or saliva requires several steps and complex equipment.
In all, our work opens new ways to extract high-quality total RNA rapidly and simply from a small quantity of blood, making the process of RNA extraction more accessible.ĭNA and RNA can be isolated from viruses, circulating tumor cells, and more to assess a patient for various pathologies.
Most notably, RNA adsorption on the beads is strong enough to counter electrophoretically-driven desorption. Our results show that DNase I and other contaminants surrounding the beads get washed away more effectively via electrophoretic transport. To improve the purification of the RNA during the bead transport through the microchannel, we also investigated the effect of a synergetic application of the electrokinetic flow. These manipulations to the conventional protocol yielded RNA amplifiable within 40 cycles of reverse transcription quantitative PCR (RT-qPCR) when using the microfluidic device to simplify the wash steps. Thus, the time to complete RNA extraction can be reduced from 2 h to 40 min. With our microfluidic method, the number of wash steps can be reduced from four to one. Our results first show that RNA integrity is maintained when the reagent volumes are scaled down by a factor of 22 and the wash buffers are combined 1:1. Here, we investigated the translation of a high volume magnetic bead-based total RNA extraction method (from human whole blood) onto a low input volume microfluidic device.
Microfluidic devices can offer the opportunity to reduce the number of hands-on steps, decrease the volumes of reagents required for purification, and make extraction high throughput. Current methods for total RNA extraction are time-consuming and require several hands-on steps and specialized equipment.
0 kommentar(er)
