A lab-on-a-chip for rapid miRNA extraction


Autoři: Ole Behrmann aff001;  Matthias Hügle aff001;  Peter Bronsert aff003;  Bettina Herde aff003;  Julian Heni aff002;  Marina Schramm aff001;  Frank T. Hufert aff001;  Gerald A. Urban aff002;  Gregory Dame aff001
Působiště autorů: Department of Microbiology and Virology, Brandenburg Medical School Fontane, Neuruppin, Germany aff001;  Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany aff002;  Institute for Surgical Pathology, Medical Center–University of Freiburg, Freiburg, Germany aff003;  Faculty of Medicine, University of Freiburg, Freiburg, Germany aff004;  German Cancer Consortium (DKTK), Partner Site Freiburg, Freiburg, Germany aff005
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226571

Souhrn

We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 μl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis.

Klíčová slova:

Elution – Gels – Heat shock response – Lysis (medicine) – Microfluidics – MicroRNAs – RNA extraction – Small nucleolar RNA


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Článek vyšel v časopise

PLOS One


2019 Číslo 12