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Photo-isolation chemistry for high-resolution and deep spatial transcriptome with mouse tissue sections.

Mizuki Honda1, Ryuichi Kimura1, Akihito Harada2

  • 1Department of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8607, Japan.

STAR Protocols
|May 2, 2022
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Summary

This study optimizes photo-isolation chemistry (PIC) for precise transcriptome analysis in mouse tissue sections. The refined protocol allows targeted gene expression profiling from specific tissue areas using UV light.

Keywords:
Gene ExpressionMolecular BiologyMolecular/Chemical ProbesRNAseqSequence analysisSequencing

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Transcriptome analysis typically requires bulk tissue, limiting spatial resolution.
  • Photo-isolation chemistry (PIC) offers a method for isolating transcriptome information from specific tissue regions.
  • Existing PIC protocols require optimization for various tissue preservation methods.

Purpose of the Study:

  • To present an optimized photo-isolation chemistry (PIC) protocol.
  • To adapt PIC for formalin-fixed frozen, paraffin-embedded, and fresh-frozen mouse tissue sections.
  • To enable targeted transcriptome analysis from defined areas within diverse animal tissues.

Main Methods:

  • Optimized tissue section preparation and permeabilization.
  • In situ reverse transcription utilizing photo-caged primers.
  • Immunostaining combined with UV-mediated uncaging for targeted cDNA synthesis.
  • Linear amplification of uncaged cDNAs, followed by library preparation and quantification.

Main Results:

  • A robust and optimized PIC protocol applicable to multiple mouse tissue section types.
  • Successful isolation of transcriptome information from specifically targeted areas.
  • Demonstrated applicability across formalin-fixed frozen, paraffin-embedded, and fresh-frozen samples.

Conclusions:

  • The optimized PIC protocol enhances spatial resolution in transcriptome analysis.
  • This method provides a valuable tool for studying gene expression in specific tissue microenvironments.
  • The protocol's versatility supports its application in various animal research models.