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Site-specific gene expression analysis from archived human intestine samples combining laser-capture microdissection
A Braun1, C Martinez1,2,3, S Schmitteckert1,3
1Department of Human Molecular Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany.
Neurogastroenterology and Motility
|December 2, 2017
Summary
This study optimized RNA isolation from specific gut layers using laser-capture microdissection and NanoString technology, revealing distinct gene expression patterns crucial for understanding gastrointestinal disorders.
Area of Science:
- Gastroenterology
- Molecular Biology
- Genomics
Background:
- Altered gene expression in intestinal wall layers is implicated in gastrointestinal disorders.
- Previous studies lacked systematic comparison of gene expression across all gut layers using fresh human samples.
Purpose of the Study:
- To develop an optimized protocol for site-specific RNA isolation from distinct intestinal layers.
- To enable high-quality RNA yield for gene expression analysis using laser-capture microdissection (LCM) and NanoString nCounter.
Main Methods:
- Utilized LCM to isolate RNA from epithelium, lamina propria, myenteric plexus, submucosa, and tunica muscularis of human colon samples.
- Employed NanoString nCounter technology for site-specific marker gene expression analysis on isolated RNA.
Main Results:
- Sufficient RNA quantity and quality were obtained from epithelial, lamina propria, and myenteric plexus layers with ~10 mm² tissue.
- ~40 mm² tissue was required for less cellular submucosal and tunica muscularis layers.
- NanoString analysis demonstrated site-specific gene expression patterns across colonic layers with high correlation (r > 0.9).
Conclusions:
- Laser-capture microdissection combined with NanoString analysis provides a sensitive and reliable method for mRNA detection and quantification.
- This approach is effective for analyzing gene expression in histologically heterogeneous tissues.
- The optimized protocol facilitates site-specific molecular profiling of the intestinal wall.
