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

PLoS One. 2019 Dec 19;14(12):e0226571. doi: 10.1371/journal.pone.0226571. eCollection 2019.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis / instrumentation*
  • Lab-On-A-Chip Devices*
  • MicroRNAs / isolation & purification*
  • RNA, Small Nucleolar / isolation & purification
  • Time Factors

Substances

  • MicroRNAs
  • RNA, Small Nucleolar

Grants and funding

The German Federal Ministry for Education and Research (BMBF, Grant no.: 031A094D) project “Isothermal Multiparametric tumor-associated μRNA Analytic (IMRA)” as well as the Ministry of Science, Research and Culture (MWFK; Grant No. 06-GeCa:H228- 05/002/008, “Multiomics” Gesundheitscampus Brandenburg) has financed this work. The article processing charge was funded by the German Research Foundation (DFG) and the University of Freiburg in the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.