Document Detail


Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis.
MedLine Citation:
PMID:  22721721     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Cynthia Xin-Zhao Wang; Kaiyan Zhang; Bogale Aredo; Hua Lu; Rafael L Ufret-Vincenty
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-06-18
Journal Detail:
Title:  Experimental eye research     Volume:  102     ISSN:  1096-0007     ISO Abbreviation:  Exp. Eye Res.     Publication Date:  2012 Sep 
Date Detail:
Created Date:  2012-09-03     Completed Date:  2012-12-04     Revised Date:  2013-09-03    
Medline Journal Info:
Nlm Unique ID:  0370707     Medline TA:  Exp Eye Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  1-9     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Ltd. All rights reserved.
Affiliation:
Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9057, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Biological Markers / metabolism
Cell Separation / methods*
Fluorescent Antibody Technique, Indirect
Mice
Mice, Inbred C57BL
Microscopy, Fluorescence
RNA / analysis*,  isolation & purification*
RNA Stability
Real-Time Polymerase Chain Reaction
Retinal Pigment Epithelium / chemistry,  cytology*,  metabolism
Transcriptome
Zonula Occludens-1 Protein / metabolism
Grant Support
ID/Acronym/Agency:
EY020799/EY/NEI NIH HHS; P30 EY020799/EY/NEI NIH HHS
Chemical
Reg. No./Substance:
0/Biological Markers; 0/Tjp1 protein, mouse; 0/Zonula Occludens-1 Protein; 63231-63-0/RNA
Comments/Corrections

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