Summary
The patch-clamp technique has allowed detailed studies on the electrical properties of neurons. Dye loading through patch pipettes has allowed characterizing the morphological properties of the neurons. In addition, the patch-clamp technique also allows harvesting mRNA from single cells to study gene expression at the single-cell level (known as single-cell reverse transcription–polymerase chain reaction [RT–PCR] [1-3]). The combination of these three approaches allows determination of the Gene expression, Electrophysiology and Morphology (GEM) profile of neurons (gene expression, electrophysiology, and morphology) using a single patch pipette and patch-clamp recording. This combination provides a powerful technique to study and correlate the neuron’s gene expression with its phenotype (electrical behavior and morphology) ( 4 – 7 ). The harvesting and amplification of single-cell mRNA for gene expression studies is a challenging task, especially for researchers with sparse or no training in molecular biology (see Notes 1 and 2). Here, we describe in detail the GEM profiling approach with special attention to the gene expression profiling.
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Acknowledgments
The authors thank Prof. Phil Goodman for helpful insights on the statistical approaches to the single-cell RT–PCR data analysis. We thank Shaoling Ma, Claudia Herzberg, Raya Eilam, and Tal Hetzroni for their technical assistance. This work was supported by the National Alliance for Autism Research and a European Union grant.
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Toledo-Rodriguez, M., Markram, H. (2007). Single-Cell RT–PCR, a Technique to Decipher the Electrical, Anatomical, and Genetic Determinants of Neuronal Diversity. In: Molnar, P., Hickman, J.J. (eds) Patch-Clamp Methods and Protocols. Methods in Molecular Biology™, vol 403. Humana Press. https://doi.org/10.1007/978-1-59745-529-9_8
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