Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

Summary

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

Abstract

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

Introduction

The RPE comprises a unique monolayer of pigmented cells located between the neural retina and the vascular choroid, and the RPE has well-recognized roles in the development and maintenance of normal retinal function, including phototransduction^1,2. More recently, the RPE has been assigned an additional key role in eye growth regulation³ and, thus, the development of myopia⁴. This assignment is based on the RPE's critical location, interposed between the retina and choroid and the now broad acceptance that eye growth and, thus, refractive errors are regul....

Protocol

All animal care and treatments used in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocols were approved by the Animal Care and Use Committee of the University of California, Berkeley.

1. Enucleation of the guinea pig eye

1. Euthanize a guinea pig with an intracardiac injection of sodium pentobarbital delivered under anesthesia (5% isoflurane in oxygen).
2. Enucleate the eyes with the aid of forceps and scissors, and immediately transfer them to a 10 cm Petri dish with sterile phosphate-buffered saline (PBS) for washing. Transfe....

Results

The analysis of the RPE samples collected using the above protocol showed well-preserved RNA (RIN >8.0, Figure 2B), with 240.2 ng ± 35.1 ng per eye (n = 8, NanoDrop, Figure 2B). To further evaluate the quality of the isolated RPE samples, particularly the absence of choroidal and scleral contaminants, we examined the expression of representative genes for each of the latter tissues in the RPE samples¹⁹. The RPE samples demonstrat.......

Discussion

In this article, we describe a method for isolating RPE, appropriate for RPE gene expression analyses, from the eyes of young, pigmented guinea pigs. The merits of this protocol are that it yields high-quality RPE samples that are relatively free from contamination, with RNA suitably preserved for RNA-specific analyses and, yet, is relatively simple and efficient. While in the example provided here, the RPE samples were collected from the eyes of a young (2 weeks old) guinea pig, the protocol has also been used.......

Materials

Name	Company	Catalog Number	Comments
1 mL Syringe with Slip Tip	Bd Vacutainer Labware Medical	22-253-260
2-Mercaptoethanol	Invitrogen	21985-023
6 Well Tissue Culture Plate with Lid, Flat Bottom, Sterile	pectrum Chemical Mfg. Corp	970-95008
12 Well Tissue Culture Plate with Lid, Individual, Sterile	Thomas Scientific LLC	1198D72
Agilent 2100 Bioanalyzer			automated electrophoresis to check RNA quality
Balanced Salt Solutions	Gibco	10010031
Bonn Micro Forceps, Straight Smooth, 0.3 mm Tip, 7 cm	Fine Science Tools, Inc.	11083-07
Dumont forceps no. 5	ROBOZ	RS-5045
Hypodermic disposable needles	Exelint International, Co.	26419
Hypodermic disposable needles	Exelint International, Co.	26437
MiniSpin Microcentrifuges	Eppendorf	540108	Max. Speed: 8,000 g
RNAlater Stabilization Solution	Invitrogen	AM7020	tissue storage reagent
RNeasy Mini kits	Qiagen	74104	RNA isolation kit
Student Vannas Spring Scissors	Fine Science Tools, Inc.	91500-09