Retinal pigment epithelium or RPE is critical for the retinal health. And studying the isolated RPE is crucial for the retinal disease. This method of RPE isolation from the guinea pig, a popular myopia model, aids in solving the challenging problem.
The main advantage of this technique is that it is relatively simple and yield high-quality RPE sample suitable for the biological molecular studies including the RPE analysis. By perfectly following the technique demonstrated and observing how to manipulate the dissecting tools, one can master this procedure quickly with some practice. After humanely euthanizing the guinea pig and enucleating the animal's eyes, immediately wash the eyes by transferring them to a 10 centimeter Petri dish containing sterile phosphate buffered saline or PBS.
After washing, transfer the eyes to fresh PBS solution. Now working under a dissecting microscope, use an 18 gauge needle to make an initial small opening in the sclera of one eye, approximately one millimeter behind the limbal boundary between the cornea and sclera. Using scissors, remove the anterior segment, including the cornea, iris, ciliary body, and crystalline lens.
Then working with a remaining posterior ocular segment, use forceps to grasp and gently tug on the zonule of Zinn, followed by detaching the retina from RPE/choroid/sclera complex, and peeling away the retina without fragmentation. After the retina has been completely removed, immerse the remaining posterior eye cup, which includes the RPE, choroid, and sclera in one of the wells of a 12-well plate containing two milliliters of tissue storage reagent and keep it immersed for five minutes. To rinse away the storage reagent, transfer the eye cup to another well filled with four milliliters of PBS for 10 seconds before moving it to a third well filled with two milliliters of PBS.
Before proceeding to the final RPE isolation step, prepare a one milliliter syringe filled with PBS and attach a 30 gauge needle. Gently push on the syringe plunger to create a jet stream of PBS. Working under a microscope, first, aim this stream of PBS at the RPE to make a small tear or hole in it.
Then direct the stream of PBS into the created opening to detach the RPE as a sheet from the choroid. After detaching the RPE from the choroid, collect the RPE cells in a needleless one milliliter syringe and transfer the collected sample to a 1.5 milliliter tube. Centrifuge the tube with the collected RPE at 8, 000 x g for one minute to obtain an RPE pellet.
For samples to be used in RNA analyses, discard the supernatant, that is the PBS solution, and replace it with 350 milliliters of lysis buffer as included in RNA isolation kits. Pipet the content up and down 20 times to mix well and preserve the quality of the sample. For long-term storage and preservation, transfer the samples to a minus 80 degree Celsius freezer.
Use an RNA isolation kit and follow the manufacturer's instructions to isolate and collect the RNA from the RPE samples before evaluating the quality of the sample via electrophoresis. In our study, the collected RPE samples showed well preserved RNA having an RNA integrity number or RIN greater than eight. The total RNA count of around 240 nanograms per eye.
The expression level of the RPE specific gene, Rpe65, was found to be significantly higher in the RPE samples than in choroid and sclera. In contrast, the RPE samples showed minimal expression of the selected choroid sclera specific gene, collagen type alpha-one, indicating the absence of choroidal and scleral contaminants in the isolated RPE samples. The most important step of the procedure is the smooth removal of the retina without leaving behind any retinal fragment.
This RPE isolation procedure warrants explanation in vitro cell culture studies with some important differences, including the choice of media for immersing the RPE cells after the isolation.
