Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis.
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 min of enucleation, and the RPE isolated within 11 min 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.
Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9057, USA.
Cynthia
C
Xin-Zhao Wang
Kaiyan
K
Zhang
Bogale
B
Aredo
Hua
H
Lu
Rafael L
RL
Ufret-Vincenty
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification*; D020871:RNA Stability; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D059467:Transcriptome; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
D015415:Biomarkers; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D012313:RNA