Laser Capture Microdissection of Paraformaldehyde-Fixed Mouse Liver Tissue for RNA Analysis.

Abstract

Laser capture microdissection (LCM) provides spatial access to specific cell populations within complex tissues through in situ visualization and isolation. To enable transcriptomic analysis of histologically defined regions in fixed tissue, a detailed LCM protocol is presented for RNA extraction from paraformaldehyde (PFA)-fixed, Optimal Cutting Temperature (OCT) compound-embedded mouse liver sections. The protocol details the identification and collection of microscale samples (approximately 1,000 cells) through a workflow encompassing tissue fixation, sucrose dehydration, OCT embedding, cryosectioning, hematoxylin staining, and laser-capture of targeted histological areas. Using this method, a high-purity RNA (A260/A280: 1.9-2.1) was obtained. The RNA integrity number (RIN) was 6.7 ± 0.9, reflecting the expected fragmentation associated with PFA fixation. However, quantitative PCR for β-actin yielded Ct values of 17-19, and RNA sequencing performed using fragmentation-optimized library preparation generated high-quality reads, with >90% of bases meeting Q20 and Q30 thresholds, confirming that the RNA is suitable for sensitive downstream analyses. Therefore, this protocol enables spatially resolved, targeted gene expression analysis by providing RNA of defined purity and integrity from specific histological regions of PFA-fixed liver tissue.