Tissue- and Species-Specific Patterns of RNA metabolism in Post-Mortem Mammalian Retina and Retinal Pigment Epithelium

Les Kallestad,Ahmad M Khalil,Krzysztof Palczewski,Seth Blackshaw

doi:10.1038/s41598-019-51379-3

Abstract

Accurate analysis of gene expression in human tissues using RNA sequencing is dependent on the quality of source material. One major source of variation in mRNA quality is post-mortem time. While it is known that individual transcripts show differential post-mortem stability, few studies have directly and comprehensively analyzed mRNA stability following death, and in particular the extent to which tissue- and species-specific factors influence post-mortem mRNA stability are poorly understood. This knowledge is particularly important for ocular tissues studies, where tissues obtained post-mortem are frequently used for research or therapeutic applications. To directly investigate this question, we profiled mRNA levels in both neuroretina and retinal pigment epithelium (RPE) from mouse and baboon over a series of post-mortem intervals. We found substantial changes in gene expression as early as 15 minutes in the mouse and as early as three hours in the baboon eye tissues. Importantly, our findings demonstrate both tissue- and species- specific patterns of RNA metabolism, by identifying a set of genes that are either rapidly degraded or very stable in both species and/or tissues. Taken together, the data from this study lay the foundation for understanding RNA regulation post-mortem and provide novel insights into RNA metabolism in the tissues of the mammalian eye.

Highlights

Gene expression is a tightly regulated process controlled by tissue-specific transcription factors, cofactors directing the binding of transcription factors, chromatin accessibility, and by various noncoding RNAs
Generation RNA sequencing (RNA-Seq) technology has enabled the characterization of gene expression in numerous human tissues and cell types, which has in turn led to better classifications of cell types within complex tissues and their functions[36,37]
One major challenge with gene expression studies of human tissues is that many investigations use tissues that were collected post-mortem[23,24,25,26,27,28,29,30,31,39] and how these findings reflect RNA expression levels of living tissues remains to be characterized[40]

Summary

Introduction

Gene expression is a tightly regulated process controlled by tissue-specific transcription factors, cofactors directing the binding of transcription factors, chromatin accessibility, and by various noncoding RNAs. Post-mortem RNA degradation in the absence of newly transcribed RNA results in the altered relative abundance of RNA levels and does not fully reflect the state of living tissues. It remains unclear how infiltration by immune cells, proximity to vascular tissue, or the complexity of different cell types within a tissue affects the transcriptome of neighboring cells in a living tissue. While this study does not attempt to characterize the macula, further studies better characterizing macular RNA will aid in our understanding of primate post-mortem retinal tissues This investigation clearly demonstrates both cell type and species-specific patterns of RNA degradation post-mortem and illustrates the need for careful interpretation of gene expression in tissues obtained post-mortem compared to living tissues

Methods

Results

Conclusion