title: Single-cell RT-PCR, a technique to decipher the electrical, anatomical, and genetic determinants of neuronal diversity.
abstract: The patch-clamp technique has allowed for detailed studies on the electrical properties of neurons. Dye loading through patch pipettes enabled characterizing the morphological properties of the neurons. In addition, the patch-clamp technique also allows for harvesting mRNA from single cells to study gene expression at the single cell level (known as single-cell RT-PCR). The combination of these three approaches makes possible the study of the 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 investigate and correlate the neuron's gene expression with its phenotype (electrical behavior and morphology). 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 1,2 and 5). Here we describe in detail the GEM profiling approach with special attention to the gene expression profiling.
authors.affiliation: Queens Medical Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2 UH, UK, maria.toledo@nottingham.ac.uk.
authors.forename: Maria
authors.initials: M
authors.lastname: Toledo-Rodriguez
authors.forename: Henry
authors.initials: H
authors.lastname: Markram
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D016591:Histocytological Preparation Techniques / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D012333:RNA, Messenger