Single-Neuron Isolation for RNA Analysis Using Pipette Capture and Laser Capture Microdissection

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.

Researchers from the University of Pennsylvania Perelman School of Medicine 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.

Pipette holder and suction line assembly. (A) Dissembled materials: (1) pipette holder without pipette; (2) plastic tubing; (3) connected stopcock; and (4) 1-mL sterile syringe. (B) Properly assembled pipette holder and suction line. (C) Pipette holder with pipette and suction line assembly setup on the inverted microscope with a Petri dish lid containing buffer and the coverslip with cells.

Pipette holder and suction line assembly. (A) Dissembled materials: (1) pipette holder without pipette; (2) plastic tubing; (3) connected stopcock; and (4) 1-mL sterile syringe. (B) Properly assembled pipette holder and suction line. (C) Pipette holder with pipette and suction line assembly setup on the inverted microscope with a Petri dish lid containing buffer and the coverslip with cells.

Lovatt D, Bell T, Eberwine J. (2015) Single-neuron isolation for RNA analysis using pipette capture and laser capture microdissection. Cold Spring Harb Protoc 2015(1):pdb.prot072439. [protocol]

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