Single-cell RNA sequencing: An overview for the ophthalmologist

Abstract

Understanding the molecular composition of pathogenic tissues is a critical step in understanding the pathophysiology of disease and designing therapeutics. First described in 2009, single cell RNA sequencing (scRNAseq) is a methodology whereby thousands of cells are simultaneously isolated into individual micro-environments that can be altered experimentally and the genome-wide RNA expression of each cell is captured. It has undergone significant technological improvement over the last decade and gained tremendous popularity. scRNAseq is an improvement over prior pooled RNA analyses which cannot identify the cellular composition and heterogeneity of a tissue of interest. This new approach offers new opportunity for new discovery, as tissue samples can now be sub-categorized into groups of cell types based on genome-wide gene expression in an unbiased fashion. As ophthalmologists, we are uniquely positioned to obtain pathologic samples from the eye for further study. ScRNAseq has already been applied in ophthalmology to characterize retinal tissue, and it may offer the key to understanding various pathological processes in the future.

Keywords: RNA sequencing; gene expression; retina surgery; retinal pathology; single cell.

Figure 1. Schematic of single-cell RNA sequencing pipeline.

A. Tissue is isolated surgically (for example vitreous fluid), transported in saline and digested to release individual cells. B. Cells are isolated into individual micro-environments through various techniques (flow cytometry shown here) and each cell is lysed to release RNA, which is then converted to cDNA and amplified. Amplified libraries (blue circle) are then submitted for next-generation sequencing and the sequence reads are aligned to the human genome. C. A matrix containing the number of reads per gene for each cell is obtained, and after normalization and dimensionality reduction, the cells are clustered into groups that are transcriptionally similar.