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

Abstract

Background: Laser capture microdissection (LCM), when combined with immunostaining (immuno-LCM) and coupled to RNA profiling, provides a means for in situ transcriptomic interrogation of complex tissues. However, preserving RNA integrity through the multiple harsh steps of immuno-LCM has proved challenging, greatly limiting the potential for high-resolution spatial analysis of global gene expression.

New method: Here, we describe a protocol whereby perfusion fixation and subsequent ex vivo post-fixation of mouse brain with paraformaldehyde, followed by protease digestion of immuno-LCM-acquired material from brain sections, allows for isolation of RNA of relatively high integrity that is amenable to RNA-seq with minimal technical variability.

Results: The individual steps of the fixation/immuno-LCM protocol were evaluated for their respective effects on RNA integrity and found not to produce significant compromise, as judged by RIN values determined using a Bioanalyzer or Tape Station. Utilizing the fixation/immuno-LCM protocol to assess gene expression from targeted brain microvascular tissue further showed high reproducibility in both qRT-PCR and RNA-seq analysis, as determined by interpolation and Pearson Correlation, respectively, with the latter detecting ∼ 22,000 genes, including those in the established blood-brain barrier transcriptome.

Comparison with existing methods: To date, there has been no detailed analysis of how fixation combined with immuno-LCM impacts RNA integrity and transcriptomic analysis.

Conclusions: The rigorous analyses performed demonstrate that paraformaldehyde fixation - which covalently cross-links RNA and protein - can be reversed without significant damage to RNA integrity, and can be combined with immuno-LCM to enable high-resolution spatial analysis of global gene expression.