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Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression
DOI:

10:54 min

February 10, 2017

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Chapters

  • 00:05Title
  • 00:59Isolation of Embryonic Midbrain
  • 02:55Dissociation and Plating of Cells
  • 05:34Harvesting Cultured Dopaminergic Neurons for RNA Sequencing
  • 07:04Generation, Purification and Amplification of First-strand cDNA
  • 09:13Results: RNA-seq Detection of Expressed Transcripts in Single Dopaminergic Neurons
  • 09:39Conclusion

Summary

Automatic Translation

In Parkinson's Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

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