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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.
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.
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 phototransduction1,2. 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....
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
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.......
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.......
This study is supported by the Japan Society for the Promotion of Science Overseas Research Fellowships (S.G.), a Loris and David Rich Postdoctoral Scholar (S.G.), and a grant from the National Eye Institute of the National Institutes of Health (R01EY012392; C.F.W.).
....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 |
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