title: Isolating dividing neural and brain tumour cells for gene expression profiling.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Griffith Health Institute, Griffith University, Southport, QLD 4222, Australia; Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
authors.forename: Berwini
authors.initials: B
authors.lastname: Endaya
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
authors.forename: Brenton
authors.initials: B
authors.lastname: Cavanagh
authors.affiliation: Griffith Health Institute, Griffith University, Southport, QLD 4222, Australia; Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
authors.forename: Faisal
authors.initials: F
authors.lastname: Alowaidi
authors.affiliation: Griffith Health Institute, Griffith University, Southport, QLD 4222, Australia; Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
authors.forename: Tom
authors.initials: T
authors.lastname: Walker
authors.affiliation: Human Adult Neural Stem Cell Facility, Nuffield Department of Surgery, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: de Pennington
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
authors.forename: Jin-Ming A
authors.initials: JM
authors.lastname: Ng
authors.affiliation: National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore.
authors.forename: Paula Y P
authors.initials: PY
authors.lastname: Lam
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
authors.forename: Alan
authors.initials: A
authors.lastname: Mackay-Sim
authors.affiliation: 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.
authors.forename: Jiri
authors.initials: J
authors.lastname: Neuzil
authors.affiliation: Griffith Health Institute, Griffith University, Southport, QLD 4222, Australia; Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia. Electronic address: a.meedeniya@griffith.edu.au.
authors.forename: Adrian C B
authors.initials: AC
authors.lastname: Meedeniya
mesh_terms: D000818:Animals; D001921:Brain / Q000502:physiology / Q000503:physiopathology; D001932:Brain Neoplasms / Q000503:physiopathology; D002478:Cells, Cultured; D057930:Click Chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives; D053595:Embryonic Stem Cells / Q000502:physiology; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005910:Glioma / Q000503:physiopathology; D006801:Humans; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D058953:Neural Stem Cells / Q000502:physiology; D055495:Neurogenesis / Q000502:physiology; D009474:Neurons / Q000502:physiology; D009831:Olfactory Mucosa / Q000502:physiology; D012313:RNA / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D012313:RNA; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
keywords: Cell division; Click chemistry; FACS; Gene transcription; Glioma; Stem cells