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Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling
10:06

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Published on: November 13, 2011

Region-specific in situ hybridization-guided laser-capture microdissection on postmortem human brain tissue coupled

René Bernard1, Sharon Burke, Ilan A Kerman

  • 1Centrum für Anatomie, Institut für Integrative Neuroanatomie, Charité Campus Mitte-Universitätsmedizin Berlin, Berlin, Germany. rbbernard@gmail.com

Methods in Molecular Biology (Clifton, N.J.)
|July 16, 2011
PubMed

Insights

This study details in situ hybridization-guided laser-capture microdissection for precise postmortem human brain tissue collection. This method ensures high-quality RNA for gene profiling and quantitative real-time polymerase chain reaction (qPCR) analysis.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Genetics

Background:

  • Postmortem human brain tissue analysis is crucial for understanding neurological disorders.
  • Preserving RNA integrity in postmortem samples is challenging but essential for accurate gene expression studies.

Purpose of the Study:

  • To describe a detailed protocol for in situ hybridization-guided laser-capture microdissection (ISH-LCM) on postmortem human brain tissue.
  • To enable precise isolation of specific brain nuclei enriched with messenger RNA (mRNA).
  • To ensure high-quality gene profiling through optimized tissue handling and RNA preservation.

Main Methods:

  • In situ hybridization (ISH) for target mRNA visualization.
  • Laser-capture microdissection (LCM) for precise tissue isolation.
  • Postmortem tissue handling protocols focused on RNA integrity.
  • mRNA amplification and gene profiling using high-density microarray chips.
  • Confirmation of gene expression using quantitative real-time polymerase chain reaction (qPCR).

Main Results:

  • Successful isolation of specific brain nuclei from postmortem human brain tissue.
  • Demonstrated enrichment of mRNA in microdissected samples.
  • High-quality RNA integrity suitable for downstream molecular analyses.
  • Reliable gene profiling data obtained through microarray and qPCR.

Conclusions:

  • ISH-guided LCM is a powerful technique for targeted molecular analysis of postmortem human brain tissue.
  • The described methods ensure the preservation of RNA integrity, critical for accurate gene expression studies.
  • This approach facilitates detailed investigation into the molecular basis of neurological conditions using human brain samples.

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