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
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2007
Materias:	Medicine R Science Q
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
_version_	1718414095038808064

Gene expression analysis of in vivo fluorescent cells.

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