Immuno-laser capture microdissection of perfusion-fixed mouse brain tissue coupled to RNA-seq

Area of Science:

• Molecular Biology
• Neuroscience
• Genomics

Background:

• Laser capture microdissection (LCM) combined with immunostaining (immuno-LCM) and RNA profiling allows in situ transcriptomic analysis of tissues.
• Preserving RNA integrity during immuno-LCM is challenging, limiting high-resolution spatial gene expression analysis.

Purpose of the Study:

• To develop and validate a protocol that preserves RNA integrity during immuno-LCM for spatial transcriptomic analysis.
• To assess the impact of fixation and immuno-LCM steps on RNA quality and subsequent gene expression analysis.

Main Methods:

• A protocol involving perfusion fixation and ex vivo post-fixation of mouse brain with paraformaldehyde.
• Protease digestion of immuno-LCM-acquired material from brain sections.
• Evaluation of RNA integrity using Bioanalyzer/Tape Station and gene expression analysis via qRT-PCR and RNA-seq.

Main Results:

• The paraformaldehyde fixation/immuno-LCM protocol maintained RNA integrity, with no significant compromise observed in RIN values.
• High reproducibility was achieved in gene expression analysis (qRT-PCR and RNA-seq) from targeted brain microvascular tissue.
• RNA-seq detected approximately 22,000 genes, including those in the blood-brain barrier transcriptome.

Conclusions:

• Paraformaldehyde fixation, which cross-links RNA and protein, can be reversed without significant RNA damage.
• The developed fixation/immuno-LCM protocol enables high-resolution spatial analysis of global gene expression with minimal technical variability.