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Isolating dividing neural and brain tumour cells for gene expression profiling
Berwini Endaya1, Brenton Cavanagh2, Faisal Alowaidi1
1Griffith Health Institute, Griffith University, Southport, QLD 4222, Australia; Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
Journal of Neuroscience Methods
|October 4, 2015
Summary
This study introduces a new method using 5-ethynyl-2-deoxyuridine (EdU) to label dividing brain cells, enabling RNA preservation for gene transcription analysis in neural and tumor cells.
Area of Science:
- Neuroscience
- Molecular Biology
- Cancer Research
Background:
- Characterizing dividing brain cells is crucial for developmental biology, stem cell research, and understanding brain cancers.
- Existing anatomical data is extensive, but gene transcription data is limited due to technical challenges.
Purpose of the Study:
- To develop a method for isolating dividing brain cells while preserving RNA for gene transcription analysis.
- To overcome the limitations of existing methods like BrdU immune-labeling, which denatures RNA.
Main Methods:
- Utilized 5-ethynyl-2-deoxyuridine (EdU) to label replicating DNA in dividing cells.
- Employed a revised click chemistry technique for fluorescent tagging and fluorescence-assisted cell sorting (FACS) for isolation.
- RNA extraction and real-time PCR were used for gene expression analysis.
Main Results:
- Demonstrated proliferation and maturation-related gene expression in neurogenic tissues using acutely and 3-day-old labeled cells.
- Observed elevated expression of marker and pathway genes in dividing cells of xenografted brain tumors compared to non-dividing cells.
Conclusions:
- The EdU labeling technique conserves RNA, unlike BrdU, allowing for gene transcription analysis.
- This method enables the study of gene transcription changes related to cell proliferation and maturation in both normal and cancerous brain cells.
