Laser Capture Microdissection-Based RNA Microsequencing Reveals Optic Nerve Crush-Related Early mRNA Alterations in Retinal Ganglion Cell Layer

Abstract

Purpose To establish a method of laser capture microdissection (LCM) and RNA microsequencing for exploring optic nerve crush (ONC)–related early mRNA alterations in retinal ganglion cell (RGC) layer. Methods An LCM protocol was developed using retinal tissue sections to obtain high-quality RNA for microsequencing. Cells in the RGC layer were collected by laser pressure catapulting (LPC) using a PALM Zeiss UV LCM system. The effect of section thickness and slide type on tissue capture success and RNA yield and the integrity after LCM were evaluated. The optimal LCM protocol was used to explore ONC-related early mRNA alterations in the RGC layer. Candidate genes were validated by real-time polymerase chain reaction of the RGC layer tissue dissected by “cut and LPC” using the same LCM system. Results We successfully established an optimal LCM protocol using 30-µm–thick retinal tissue sections mounted on glass slides and laser pressure catapulting (LPC) to collect cells in the RGC layer and to obtain high-quality RNA for microsequencing. On the basis of our protocol, we identified 8744 differentially expressed genes that were involved in ONC-related early mRNA alterations in the RGC layer. Candidate genes included Atf3, Lgals3, LOC102551701, Plaur, Tmem140, and Maml1. Conclusions The LCM-based single-cell RNA sequencing allowed a new sight into the early mRNA changes of RGCs highlighting new molecules associated to ONC. Translational Relevance This technique will be helpful for more accurate transcriptome analysis of clinical pathological samples of ophthalmology and provide important reference for the discovery of new pathological diagnosis indicators and drug development targets.

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“…This knowledge could guide the development of new therapeutic strategies that inhibit both pain and cancer dissemination. Implementation of single-cell RNA-sequencing and laser-capture microdissection, or the combination of these techniques, as recently reported for optic nerve crush-related early mRNA alterations in retinal ganglion cells 9. This approach can aid to disentangle tumor from nerves signals and assist to uncover subtype-specific vulnerabilities in the PDAC/nerves relationships that could be targeted by different drugs.…”