A microfluidic approach for high efficiency extraction of low molecular weight RNA. 2010

Paul Vulto, and Gregory Dame, and Urban Maier, and Solomzi Makohliso, and Susann Podszun, and Peter Zahn, and Gerald A Urban
Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität Freiburg, Georges-Köhler-Allee 103, Freiburg, Germany. p.vulto@gmx.net

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.

UI MeSH Term Description Entries
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D002458 Cell Fractionation Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS. Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D004563 Electrochemistry The study of chemical changes resulting from electrical action and electrical activity resulting from chemical changes. Electrochemistries
D004867 Equipment Design Methods and patterns of fabricating machines and related hardware. Design, Equipment,Device Design,Medical Device Design,Design, Medical Device,Designs, Medical Device,Device Design, Medical,Device Designs, Medical,Medical Device Designs,Design, Device,Designs, Device,Designs, Equipment,Device Designs,Equipment Designs
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005591 Chemical Fractionation Separation of a mixture in successive stages, each stage removing from the mixture some proportion of one of the substances, for example by differential solubility in water-solvent mixtures. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Fractionation, Chemical,Chemical Fractionations,Fractionations, Chemical
D012329 RNA, Bacterial Ribonucleic acid in bacteria having regulatory and catalytic roles as well as involvement in protein synthesis. Bacterial RNA
D013048 Specimen Handling Procedures for collecting, preserving, and transporting of specimens sufficiently stable to provide accurate and precise results suitable for clinical interpretation. Specimen Collection,Collection, Specimen,Collections, Specimen,Handling, Specimen,Handlings, Specimen,Specimen Collections,Specimen Handlings
D044085 Microfluidics The study of fluid channels and chambers of tiny dimensions of tens to hundreds of micrometers and volumes of nanoliters or picoliters. This is of interest in biological MICROCIRCULATION and used in MICROCHEMISTRY and INVESTIGATIVE TECHNIQUES. Microfluidic
D019544 Equipment Failure Analysis The evaluation of incidents involving the loss of function of a device. These evaluations are used for a variety of purposes such as to determine the failure rates, the causes of failures, costs of failures, and the reliability and maintainability of devices. Materials Failure Analysis,Prosthesis Failure Analysis,Analysis, Equipment Failure,Analysis, Materials Failure,Analysis, Prosthesis Failure,Analyses, Equipment Failure,Analyses, Materials Failure,Analyses, Prosthesis Failure,Equipment Failure Analyses,Failure Analyses, Equipment,Failure Analyses, Materials,Failure Analyses, Prosthesis,Failure Analysis, Equipment,Failure Analysis, Materials,Failure Analysis, Prosthesis,Materials Failure Analyses,Prosthesis Failure Analyses

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