FISH and chips: a review of microfluidic platforms for FISH analysis

Abstract

Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory. Keywords Microfluidics-assisted FISH • µFISH • Microfluidics • Lab-on-a-Chip (LOC) • Fluorescence in situ hybridization (FISH) Edited by Volkhard A. J. Kempf.

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Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics

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Long Non-Coding RNAs: The Regulatory Mechanisms, Research Strategies, and Future Directions in Cancers

“…Fluorescence in situ hybridization (FISH) is a method often used to identify the cellular localization of the candidate lncRNAs. The principle is that the foreign nucleic acid containing radioactive labels (3H, 32P, 35S, 125I) or nonradioactive labels [biotin, digoxin, horseradish peroxidase, fluorescein (FITC, rhodamine)] is complementary paired with the DNA or RNA to be tested on tissues, cells or chromosomes to form a specific nucleic acid hybrid molecule (82,83). In addition to FISH localization, certain prediction software such as LncATLAS (84) can predict the cellular localization of lncRNAs; however, the predictive data need to be verified experimentally.…”