RNA Isolation from Mouse Ocular Lens Epithelium and Fiber Cell Bulk Masses

The lens is a specialized, transparent tissue in the anterior chamber of the eye that is comprised of two major cell types, epithelial cells and fiber cells. A monolayer of lens epithelial cells covers the anterior hemisphere of the lens, while most of the lens is composed of a mass of lens fiber cells. A collagenous basement membrane, known as the capsule, surrounds and encapsulates the entire tissue. Lens epithelial cells are strongly adhered to the lens capsule and can easily be separated from the bulk fiber mass by peeling the capsule away from the tissue. Difficulties obtaining sufficient RNA concentration from the low number of epithelial cells in the lens monolayer has previously impeded the study of epithelial cell vs. fiber cell transcriptomes. This protocol presents a method to cleanly separate and isolate the epithelial and fiber cell compartments and RNA concentration steps to allow subsequent transcriptomic experiments on epithelial cell samples from a single pair of mouse lenses. The ability to investigate the major cell types of the lens individually aids in investigating biological mechanisms of lens maintenance and dysregulation, enabling the characterization of cell-type-specific disruptions responsible for age-related lens pathologies.