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Related Concept Videos

FISH - Fluorescent In-situ Hybridization02:07

FISH - Fluorescent In-situ Hybridization

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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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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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In-situ Hybridization02:31

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In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
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Multiplexed in situ RNA imaging by combFISH.

Yanxiu Liu1,2, Jiayu Chen1,2, Chen Lin3,4

  • 1School of Medicine, Huaqiao University, Xiamen, 361021, Fujian, China.

Analytical and Bioanalytical Chemistry
|May 22, 2024
PubMed
Summary

We developed a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay for multiplexed RNA detection. This method enables high-throughput spatial gene expression profiling in tissues without sequencing-by-ligation chemistry.

Keywords:
RNA in situ hybridizationRolling circle amplificationSingle-cell analysisSpatial transcriptomics

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Multiplexed in situ RNA imaging is crucial for high-throughput spatial gene expression profiling.
  • Existing methods often involve complex protocols, limiting their widespread adoption.

Purpose of the Study:

  • To present a novel cyclic combinatorial fluorescent in situ hybridization (combFISH) assay.
  • To enable multiplexed RNA detection in cell cultures and tissues with a simplified protocol.

Main Methods:

  • Developed a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay.
  • Utilized rolling circle amplicons generated from a padlock probe assay.
  • Employed combinatorial detection probes for cyclic interrogation of barcode sequences.

Main Results:

  • Successfully demonstrated multiplexed RNA detection in both cell cultures and tissue sections.
  • The combFISH assay works with fresh-frozen and formalin-fixed paraffin-embedded tissues.
  • The method theoretically allows detection of 64 genes in three hybridization cycles.

Conclusions:

  • The combFISH assay offers a straightforward and efficient method for multiplexed in situ RNA imaging.
  • Elimination of sequencing-by-ligation chemistry simplifies the protocol.
  • The method has potential for broad adoption in research and clinical settings.