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FISH - Fluorescent In-situ Hybridization

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...

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Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

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Published on: December 21, 2017

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ

Jeffrey R Moffitt¹, Junjie Hao², Guiping Wang²

¹Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138; zhuang@chemistry.harvard.edu lmoffitt@mcb.harvard.edu.

Proceedings of the National Academy of Sciences of the United States of America

|September 15, 2016

Summary

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) now offers significantly higher throughput for single-cell transcriptomics. This advancement enables rapid RNA profiling in over 100,000 cells, preserving spatial context.

Keywords:

fluorescence in situ hybridization multiplexed imaging single-cell analysis transcriptomics

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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

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Published on: August 3, 2011

Area of Science:

Molecular Biology
Genomics
Cell Biology

Background:

Image-based single-cell transcriptomics complements RNA sequencing by preserving spatial context.
Current image-based methods, like MERFISH, have limitations in throughput for large-scale studies.

Purpose of the Study:

To significantly enhance the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH).
To enable large-scale RNA profiling in human cells while maintaining spatial information.

Main Methods:

Implemented chemical cleavage for signal removal instead of photobleaching in MERFISH.
Expanded the imaging field of view and incorporated multicolor imaging.
Utilized combinatorial labeling and sequential single-molecule fluorescence in situ hybridization (smFISH) for RNA identification.

Main Results:

Achieved a two-orders-of-magnitude increase in MERFISH throughput.
Successfully profiled RNA in over 100,000 human cells.
Demonstrated the ability to measure up to 40,000 cells within an 18-hour period.

Conclusions:

The enhanced MERFISH technique dramatically increases throughput for image-based single-cell transcriptomics.
This advancement expands the scope of biological questions addressable with spatial transcriptomics.
High-throughput MERFISH provides a powerful tool for analyzing RNA expression and localization in large cell populations.