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

Cynthia Xin-Zhao Wang, Kaiyan Zhang, Bogale Aredo, Hua Lu, Rafael L. Ufret-Vincenty

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalExperimental Eye Research
Volume102
DOIs
StatePublished - Sep 2012

Fingerprint

Cell Separation
RNA
Retinal Diseases
Transcriptome
Blood Vessels

Keywords

  • Gene expression
  • Mouse
  • Novel technique
  • RNA extraction
  • RPE isolation

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis. / Xin-Zhao Wang, Cynthia; Zhang, Kaiyan; Aredo, Bogale; Lu, Hua; Ufret-Vincenty, Rafael L.

In: Experimental Eye Research, Vol. 102, 09.2012, p. 1-9.

Research output: Contribution to journalArticle

Xin-Zhao Wang, Cynthia ; Zhang, Kaiyan ; Aredo, Bogale ; Lu, Hua ; Ufret-Vincenty, Rafael L. / Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis. In: Experimental Eye Research. 2012 ; Vol. 102. pp. 1-9.
@article{414f05c6bddf4463baf1e695276c0b50,
title = "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 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.",
keywords = "Gene expression, Mouse, Novel technique, RNA extraction, RPE isolation",
author = "{Xin-Zhao Wang}, Cynthia and Kaiyan Zhang and Bogale Aredo and Hua Lu and Ufret-Vincenty, {Rafael L.}",
year = "2012",
month = "9",
doi = "10.1016/j.exer.2012.06.003",
language = "English (US)",
volume = "102",
pages = "1--9",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis

AU - Xin-Zhao Wang, Cynthia

AU - Zhang, Kaiyan

AU - Aredo, Bogale

AU - Lu, Hua

AU - Ufret-Vincenty, Rafael L.

PY - 2012/9

Y1 - 2012/9

N2 - 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.

AB - 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.

KW - Gene expression

KW - Mouse

KW - Novel technique

KW - RNA extraction

KW - RPE isolation

UR - http://www.scopus.com/inward/record.url?scp=84865590817&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84865590817&partnerID=8YFLogxK

U2 - 10.1016/j.exer.2012.06.003

DO - 10.1016/j.exer.2012.06.003

M3 - Article

VL - 102

SP - 1

EP - 9

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

ER -