An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection
RNA sequencing (RNA-seq) coupled with laser capture microdissection (LCM) is a powerful tool for transcriptomic analysis in unfixed fresh-frozen tissues. Fixation of ocular tissues for immunohistochemistry commonly involves the use of paraformaldehyde (PFA) followed by embedding in Optimal Cutting Temperature (OCT) medium for long-term cryopreservation. However, the quality of RNA derived from such archival PFA-fixed/OCT-embedded samples is often compromised, limiting its suitability for transcriptomic studies. In this study, we aimed to develop a methodology to extract high-quality RNA from PFA-fixed canine eyes by utilizing LCM to isolate retinal tissue. We demonstrate the efficacy of an optimized LCM and RNA purification protocol for transcriptomic profiling of PFA-fixed retinal specimens. We compared four pairs of canine retinal tissues, where one eye was subjected to PFA-fixation prior to OCT embedding, while the contralateral eye was embedded fresh frozen (FF) in OCT without fixation. Since the mRNA obtained from PFA-fixed retinas were contaminated with genomic DNA, we employed two rounds of DNase I treatment to obtain RNA suitable for RNA-seq. Notably, the quality of sequencing reads and gene sets identified from both PFA-fixed and FF tissues were nearly identical. In summary, our study introduces an optimized workflow for transcriptomic profiling from PFA-fixed archival retina. This refined methodology paves the way for improved transcriptomic analysis of preserved ocular tissue, bridging the gap between optimal sample preservation and high-quality RNA data acquisition. Overall design: To compare the transcriptomic profile between fresh frozen and PFA-fixed canine retinal tissues, bulk RNA sequencing was performed with four biological replicates.