Microfluidic Approaches to Fluorescence In Situ Hybridization (FISH) for Detecting RNA Targets in Single Cells
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 small-molecule 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].
KeywordsLab on a chip Molecular diagnostics Gene expression Genetic analysis Single-cell analysis Nucleic acid hybridization Flow cytometry
The authors acknowledge support from Sandia National Laboratories’ Laboratory Directed Research and Development (LDRD) program. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the US Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000.
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