Single-cell RT-PCR, a technique to decipher the electrical, anatomical, and genetic determinants of neuronal diversity.
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.
Toledo-Rodriguez
Maria
M
Brain and Mind Institute, EPFL, Switzerland.
Markram
Henry
H
D000818:Animals; D004553:Electric Conductivity; D005786:Gene Expression Regulation; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D051637:Shaker Superfamily of Potassium Channels
D051637:Shaker Superfamily of Potassium Channels