Research PaperA method for isolating cortical interneurons sharing the same birthdays for gene expression studies

- Cortical interneurons sharing the same birthdates can be isolated by FACs sorting.
- Optimized EdU detection gives good signal and preserves cellular RNA integrity.
- PFA fixation improves concurrent GFP signal detection in two-colour cell sorting of EdU-labelled interneurons.
- RNA extracted from GFP+/EdU+ and GFP−/EdU+ cells can be used for qPCR analysis.

The two neuronal populations in the cortex, pyramidal neurons and interneurons, can be separated based on neurotransmitter identity, however, within this segregation a large degree of diversity exists. Investigations into the molecular diversity of neurons are impeded by the inability to isolate cell populations born at different times for gene expression analysis. Developing interneurons may be distinguished by the expression of Glutamic Acid Decarboxylase-67 (GAD67). Neuronal birthdating using nucleoside analogs is an effective means of identifying coetaneous interneurons. Using these two features, neurotransmitter identity and birthdating, we have developed a method to isolate migrating interneurons using fluorescent-activated cell sorting (FACS) for RNA extraction and gene expression analysis. We utilized 5-ethynyl-2′-deoxyuridine (EdU) to birthdate interneuron cohorts and the GAD67 knock-in GFP transgenic mice to identify interneurons. In combination, we achieved simultaneous detection of GFP and EdU signals during FACS sorting of coetaneous interneurons with minimum loss of RNA integrity. RNA quality was deemed to be satisfactory by quantitative polymerase chain reaction (qPCR) for the interneuron-specific transcript Gad67.