Comparison of Transcript and Protein Abundance in Human Donor Retinas Support Extending Postmortem Interval From 12...

To evaluate whether extending the postmortem interval (PMI) from 12 to 18 hours affects transcript and protein abundances in the human retina.

Donor eyes were recovered from postmortem donors (n = 7 pairs) and stored at 2°C-8°C. Only donor eyes with ocular cooling within eight hours of death were examined. The OD retina was preserved at 12 hours and the OS retina at 18 hours postmortem. Retinas were flash frozen with liquid nitrogen. The macular region superior to the fovea was used for RNA sequencing (RNA-seq), whereas the inferior region was processed for tandem mass tag (TMT) quantitative proteomics. RNA library preparations were performed simultaneously, and the sequencing of all samples were conducted on the same chip. Proteins were isolated and labeled with 16-plex TMTPro isobaric tags, and all samples were analyzed in a 20-fraction TMT-mass spectrometry experiment. RNA-seq data were processed using DESeq2 (Bioconductor) and proteomic data were analyzed using the previously published PAW pipeline.

RNA-seq identified 22,446 transcripts, with only five transcripts showing significant differential expression (adjusted P < 0.1 and a Log2FoldChange > 1) between 12 and 18 hours. No enriched pathways were associated with these changes. Proteomic analysis identified 6,109 proteins, with no significant differences in abundance (all adjusted P > 0.05). Retinal cell-type specific markers and markers relevant for disease research, such as age-related macular degeneration and glaucoma, remained stable across both time points.

Extending the PMI cutoff from 12 to 18 hours did not significantly impact transcriptomic or proteomic integrity in human donor retinas. This PMI extension greatly increases the availability of donor eyes for biomedical research.