Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis

Abstract

RPE cells are involved in the pathogenesis of many retinal diseases. Accurate analysis of RPE gene expression profiles in different scenarios will increase our understanding of disease mechanisms. Our objective in this study was to develop an improved method for the isolation of RPE cells, specifically for RNA analysis. Mouse RPE cells were isolated using different techniques, including mechanical dissociation techniques and a new technique we refer to here as “Simultaneous RPE cell Isolation and RNA Stabilization” (SRIRS method). RNA was extracted from the RPE cells. An RNA bioanalyzer was used to determine the quantity and quality of RNA. qPCR was used to determine contamination with non-RPE-derived RNA. Several parameters with a potential impact on the isolation protocol were studied and optimized. A marked improvement in the quantity and quality of RPE-derived RNA was obtained with the SRIRS technique. We could get the RPE in direct contact with the RNA protecting agent within 1 minute of enucleation, and the RPE isolated within 11 minutes of enucleation. There was no significant contamination with vascular, choroidal or scleral-derived RNA. We have developed a fast, easy and reliable method for the isolation of RPE cells that leads to a high yield of RPE-derived RNA while preserving its quality. We believe this technique will be useful for future studies looking at gene expression profiles of RPE cells and their role in the pathophysiology of retinal diseases.

Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery

“…Thus, our finding mRNA for RPE65 in the choroid samples is not indicative of contamination from RPE cells. Although, in fresh tissue, retina is easily separated from RPE, which adheres to the choroid (Bruch’s membrane), treatment with RNAlater causes the RPE to adhere tightly to the retina (Malik et al., 2003; Wang et al, 2012). These factors, along with our observation during tissue dissection that retina/RPE together cleanly separated from the choroid lead us to conclude that there was little, if any, RPE contamination of the choroidal samples.…”

“…Extraocular muscles, conjunctiva, and orbital fat were trimmed from the exterior surface of the eye and the cornea dissected away just behind the corneoscleral junction. While viewing through a surgical microscope, the lens and vitreous humor were removed; the retina and RPE, which were tightly bound to each other (Malik et al, 2003; Wang et al, 2012), were then lifted from the eyecup. While still immersed in RNAlater, choroid was teased from the scleral inner surface by gentle separation using the rounded ends of forceps, collected, and then frozen in liquid nitrogen.…”

Systemic treatment with a 5HT1a agonist induces anti-oxidant protection and preserves the retina from mitochondrial oxidative stress

“…It was then transferred to a tube containing 200 μl of RNAprotect cell reagent (QIAGEN) and incubated for 10 min at room temperature. The tube was agitated to release RPE cells, as described in (Xin-Zhao Wang, Zhang, Aredo, Lu and Ufret-Vincenty, 2012). The eye cup was removed.…”

An easy, rapid method to isolate RPE cell protein from the mouse eye

“…Due to the strong adhesion of the RPE to Bruch's membrane and choroid, and the thin Bruch's membrane due to the small globe size, prior attempts using mechanical debridement to isolate the RPE from choroid have been challenging and unsuccessful at obtaining pure populations of RPE cells(Xin-Zhao Wang et al, 2012). Therefore, we attempted a strategy that did not depend upon mechanical manipulation.…”

“…This technique appears to be valuable for investigating subcellular organelles. Along with the work of Wang et al(Xin-Zhao Wang et al, 2012), who used enzymatic digestion to extract high quality RNA, we hope that our innovation will provide a valuable method for recovering RPE proteins that will improve our ability to study RPE cell behavior in both health and disease.…”