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.

Eskitis Institute for Drug Discovery Griffith University Nathan QLD 4111 Australia

Griffith Health Institute Griffith University Southport QLD 4222 Australia; Eskitis Institute for Drug Discovery Griffith University Nathan QLD 4111 Australia

Griffith Health Institute Griffith University Southport QLD 4222 Australia; Institute of Biotechnology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague 4 Krc Czech Republic

Human Adult Neural Stem Cell Facility Nuffield Department of Surgery John Radcliffe Hospital University of Oxford Oxford OX3 9DU United Kingdom

National Cancer Centre Singapore 11 Hospital Drive Singapore 169610 Singapore

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DNA Replication: From Radioisotopes to Click Chemistry