A microfluidic approach for high efficiency extraction of low molecular weight RNA

Abstract

The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a factor of 20 with respect to commercial extraction kits. The presented solution may find broad acceptance and application in drug discovery and clinical diagnostics.

A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction

“…The use of magnetic beads, 12,13 membrane based extraction 2,14 or paperuidics 15 are some examples. In previous works, we have presented on-chip lysis and RNA extraction from bacteria 16,17 as well as FFE preconcentration of viable bacteria from liquid samples. 18,19 Here, we present a lab-on-a-chip system based on these results.…”

“…It is performed until the formation of heat induced gas bubbles is observed. Parameters for lysis are established by Vulto et al 16 The devices used for lysis are a function generator (33120A, Hewlett-Packard) and a voltage amplier (A600, FLC Electronics).…”

In-Situ Electrophoretic Mobility Determination by Particle Image Velocimetry for Efficient Microfluidic Enrichment of Bacteria

“…Environmental samples often contain low amounts of bacteria as well as substances that inhibit PCR, necessitating preprocessing of the raw sample. In former work, both the enrichment of bacteria from suspension as well as size-selective purification of target nucleic acids on microfluidic platforms have been demonstrated [1,2].…”

Impact of microfluidic processing on bacterial ribonucleic acid expression

“…2 However, these systems performed only the detection stage of the on-chip process. Microfluidics has also been utilised for on-chip extraction of viral RNA in blood 12 and for bacterial RNA extraction, 13 but the RNA expression was not benchmarked against traditional processes. A major challenge in using RNA expression lies in processing and extracting, in a robust and repeatable manner, RNA from a patient sample before the occurrence of any changes in RNA expression.…”