Microfluidic Approaches to Fluorescence In Situ Hybridization (FISH) for Detecting RNA Targets in Single Cells

Abstract

Fluorescence in situ hybridization (FISH) is a powerful molecular technique in cell biology and microbiology for detection and localization of a nucleic acid target within an intact cell or chromosome spread, based on hybridization of a fluorescently labeled nucleic acid "probe" to its complementary target. In some instances, FISH analysis is performed on intact samples-whether thin tissue sections, or environmental samples, allowing the nucleic acid target to be localized in context with other cells. FISH evolved from in situ hybridization (ISH) techniques utilizing radiolabeled probes. By comparison, FISH typically utilizes smallmolecule fluorophores. This labeling approach eliminates the hazards associated with radioactivity, and allows analysis with common laboratory instrumentation, including epifluorescence or laser-scanning confocal microscopes, or flow cytometers. Unlike PCR, sequencing, or most other nucleic acid analysis methods, FISH is fundamentally a single-cell measurement technique. Whether using imaging or flow cytometry as a readout, the signals from FISH are detected and analyzed on a cell-by-cell basis, affording a unique capability for studying rare events or heterogeneity within a population. Coupling of FISH with flow sorters also affords a unique capability for enriching cell or chromosome populations or even isolating single cells based on presence of specific nucleic acid targets [7,10,43].

Microfluidics as a Novel Tool for Biological and Toxicological Assays in Drug Discovery Processes: Focus on Microchip Electrophoresis

“…Approximately 10 years later, Manz and co-workers published a paper describing the use of micromachining to build up a miniaturized total chemical analysis system, laying the foundations for the development of an integrated microfluidic system using capillary electrophoresis (CE) [3]. Since then, the number and types of microfluidic-based technologies have dramatically increased and they have been applied in a number of fields including chemistry, biochemistry, physics, biology, and molecular biology [4][5][6][7]. The increased interest of the research community for these technologies is supported by thousands of publications in peer-reviewed journals and conference proceedings [8].…”

An Examination of the Validity of MRPS Method for the Detection of Label-free E. Coli and Enterococci in Ships Ballast Water

“…The quality of ship's ballast water will be accessed before discharging and it becomes a technical challenge. The most well-known methods of detection for indicative microorganisms sample from ships ballast water are Adenosine triphosphate (ATP) [6][7][8] [22,23], fluorescent in-situ hybridization (FISH) [24,25] and surface enhanced Raman scattering (SERS) [26,27]. Some of their techniques can detect single cell with high accuracy.…”