A microfluidic approach for high efficiency extraction of low molecular weight RNA.
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.
Vulto
Paul
P
Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität Freiburg, Georges-Köhler-Allee 103, Freiburg, Germany. p.vulto@gmx.net
Dame
Gregory
G
Maier
Urban
U
Makohliso
Solomzi
S
Podszun
Susann
S
Zahn
Peter
P
Urban
Gerald A
GA
D002458:Cell Fractionation / Q000295:instrumentation*; D005591:Chemical Fractionation / Q000295:instrumentation*; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000235:genetics*; D044085:Microfluidics / Q000295:instrumentation*; D008970:Molecular Weight; D012329:RNA, Bacterial / Q000302:isolation & purification*; D013048:Specimen Handling / Q000295:instrumentation*
D012329:RNA, Bacterial