title: Single-neuron isolation for RNA analysis using pipette capture and laser capture microdissection.
abstract: The field of single-cell analysis has greatly benefitted from recent technological advances allowing scientists to study genomes, transcriptomes, proteomes, and metabolomes at the single-cell level. Transcriptomics allows a unique window into cell function and is especially useful for studying global variability among single cells of seemingly the same type. Generating transcriptome data from RNA samples has become increasingly easy and can be done using either microarray or RNA-Seq techniques. RNA isolation is the first step of transcriptomics. Numerous RNA isolation procedures exist and differ with respect to the type and number of cells from which they are capable of isolating RNA. Although it is trivial to isolate RNA from bulk tissue or culture plates, sophisticated methods are required to capture RNA from single cells in a pool of cells or in intact tissue. We describe here the protocols used for isolating the soma of single neurons in cultures and in tissue slices using the pipette capture and the PALM or laser capture microdissection (LCM) approaches, respectively. LCM was developed to isolate cells from tissue sections primarily for pathological tissue analysis. LCM can be used to isolate individual cells or groups of cells from ethanol or paraffin-embedded formalin-fixed tissue sections and dissociated tissue cultures. The soma isolates from either technique can subsequently be used for RNA amplification procedures and transcriptome analysis. These procedures can also be adapted to other cell types in cultures and tissue sections and can be used on neuronal subcellular structures, such as dendrites.
authors.affiliation: Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104.
authors.forename: Ditte
authors.initials: D
authors.lastname: Lovatt
authors.affiliation: Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bell
authors.affiliation: Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104.
authors.forename: James
authors.initials: J
authors.lastname: Eberwine
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology; D061346:Laser Capture Microdissection; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D012313:RNA / Q000032:analysis* / Q000235:genetics
chemical_list: D012313:RNA