An investigation into simplifying total RNA extraction with minimal equipment using a low volume, electrokinetically driven microfluidic protocol

Abstract

Current methods for total RNA extraction are time-consuming and require several hands-on steps and specialized equipment. 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. 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. 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. With our microfluidic method, the number of wash steps can be reduced from four to one. Thus, the time to complete RNA extraction can be reduced from 2 h to 40 min. 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. 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. Our results show that DNase I and other contaminants surrounding the beads get washed away more effectively via electrophoretic transport. Most notably, RNA adsorption on the beads is strong enough to counter electrophoretically-driven desorption. 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.