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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...
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Related Experiment Video

Updated: May 31, 2025

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection
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Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

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Novel photocrosslinking chemical probes utilized for high-resolution spatial transcriptomics.

Leslie Spitalny1, Natalie Falco1, Whitney England1

  • 1Department of Pharmaceutical Sciences, University of California Irvine California 92697 USA rspitale@uci.edu.

RSC Chemical Biology
|January 23, 2025
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Summary

Researchers developed CrossSeq, a new spatial transcriptomic analysis method. This technique uses photocrosslinking probes to precisely isolate cells for RNA sequencing, advancing our understanding of tissue microenvironments.

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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
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Area of Science:

  • Cell biology
  • Molecular biology
  • Genomics

Background:

  • Cellular functions are optimized within complex tissue microenvironments through specific RNA expression.
  • Spatial transcriptomics merges cell tagging with RNA sequencing for understanding spatial RNA expression.
  • Current methods face limitations in spatial resolution, RNA sequencing capacity, and multiplexing for distinct cell isolation.

Purpose of the Study:

  • To develop a novel method, CrossSeq, for multiplexed spatial transcriptomic analysis.
  • To overcome limitations of existing spatial transcriptomic techniques regarding resolution, RNA capacity, and multiplexing.
  • To enable precise isolation of spatially-distinct cells within tissue landscapes for detailed analysis.

Main Methods:

  • CrossSeq utilizes photocrosslinking fluorescent probes and confocal microscopy for region demarcation.
  • Investigated phenyl azide and diazirine crosslinking scaffolds, defining their photoactivity.
  • Deployed an aryl azide scaffold with three fluorophores for multiplexing on glyoxal-fixed cells, followed by flow cytometry analysis.

Main Results:

  • Successfully demonstrated the capability of CrossSeq to demarcate user-defined regions of interest on fixed cells.
  • Defined photoactivity profiles for phenyl azide and diazirine crosslinking scaffolds.
  • Applied CrossSeq to a metastatic cancer cell model to analyze gene expression differences between migratory and exterior cell populations.

Conclusions:

  • CrossSeq offers a valuable new tool for spatial transcriptomic analysis.
  • The technology enables easier access to spatial transcriptomic data using conventional microscopy.
  • CrossSeq facilitates the investigation of gene expression dynamics in specific cellular locations within complex biological systems.