Isolating dividing neural and brain tumour cells for gene expression profiling

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F16%3A00459241" target="_blank" >RIV/86652036:_____/16:00459241 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.jneumeth.2015.09.020" target="_blank" >http://dx.doi.org/10.1016/j.jneumeth.2015.09.020</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jneumeth.2015.09.020" target="_blank" >10.1016/j.jneumeth.2015.09.020</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Isolating dividing neural and brain tumour cells for gene expression profiling

Popis výsledku v původním jazyce

Background: The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. New method: We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. Results: 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. Comparison with existing method: BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. Conclusions: The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation. (C) 2015 Elsevier B.V. All rights reserved.

Název v anglickém jazyce

Isolating dividing neural and brain tumour cells for gene expression profiling

Popis výsledku anglicky

Background: The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. New method: We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. Results: 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. Comparison with existing method: BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. Conclusions: The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation. (C) 2015 Elsevier B.V. All rights reserved.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

EB - Genetika a molekulární biologie

OECD FORD obor

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Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2016

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Neuroscience Methods

ISSN

0165-0270

e-ISSN

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Svazek periodika

257

Číslo periodika v rámci svazku

JAN 15 2016

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

13

Strana od-do

121-133

Kód UT WoS článku

000366224100012

EID výsledku v databázi Scopus

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