Gene expression analysis of in vivo fluorescent cells.

<h4>Background</h4>The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Micr...

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Autores principales: Konstantin Khodosevich, Dragos Inta, Peter H Seeburg, Hannah Monyer
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Publicado: Public Library of Science (PLoS) 2007
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Acceso en línea:https://doaj.org/article/3876c03255c04da88840f09d5d9b9e2c
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spelling oai:doaj.org-article:3876c03255c04da88840f09d5d9b9e2c2021-11-25T06:10:31ZGene expression analysis of in vivo fluorescent cells.1932-620310.1371/journal.pone.0001151https://doaj.org/article/3876c03255c04da88840f09d5d9b9e2c2007-11-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0001151https://doaj.org/toc/1932-6203<h4>Background</h4>The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.<h4>Methodology/principal findings</h4>We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of approximately 2ratio1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).<h4>Conclusions/significance</h4>Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.Konstantin KhodosevichDragos IntaPeter H SeeburgHannah MonyerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 2, Iss 11, p e1151 (2007)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Konstantin Khodosevich
Dragos Inta
Peter H Seeburg
Hannah Monyer
Gene expression analysis of in vivo fluorescent cells.
description <h4>Background</h4>The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.<h4>Methodology/principal findings</h4>We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of approximately 2ratio1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).<h4>Conclusions/significance</h4>Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.
format article
author Konstantin Khodosevich
Dragos Inta
Peter H Seeburg
Hannah Monyer
author_facet Konstantin Khodosevich
Dragos Inta
Peter H Seeburg
Hannah Monyer
author_sort Konstantin Khodosevich
title Gene expression analysis of in vivo fluorescent cells.
title_short Gene expression analysis of in vivo fluorescent cells.
title_full Gene expression analysis of in vivo fluorescent cells.
title_fullStr Gene expression analysis of in vivo fluorescent cells.
title_full_unstemmed Gene expression analysis of in vivo fluorescent cells.
title_sort gene expression analysis of in vivo fluorescent cells.
publisher Public Library of Science (PLoS)
publishDate 2007
url https://doaj.org/article/3876c03255c04da88840f09d5d9b9e2c
work_keys_str_mv AT konstantinkhodosevich geneexpressionanalysisofinvivofluorescentcells
AT dragosinta geneexpressionanalysisofinvivofluorescentcells
AT peterhseeburg geneexpressionanalysisofinvivofluorescentcells
AT hannahmonyer geneexpressionanalysisofinvivofluorescentcells
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