Laser capture microdissection of single neurons with morphological visualization using fluorescent proteins fused to transmembrane proteins.

Chang, Ching Ching; Chong, Hai Tarng; Tashiro 田代歩, Ayumu

Gene expression analysis in individual neuronal types helps understand brain function. Genetic methods expressing fluorescent proteins are widely used to label specific neuronal populations. However, because cell type specificity of genetic labeling is often limited, it is advantageous to combine genetic labelling with additional methods to select specific cell/neuronal types. Laser capture microdissection is one of such techniques with which one can select a specific cell/neuronal population based on morphological observation. However, a major issue is disappearance of fluorescence signals during tissue processing required for high quality sample preparation. Here, we developed a simple, novel method in which fluorescence signals are preserved. We use genetic labeling with fluorescence proteins fused to transmembrane proteins, which shows highly stable fluorescence retention and allows for the selection of fluorescent neurons/cells based on morphology. Using this method in mice, we laser-captured neuronal somata and successfully isolated RNA. We determined that ∼100 cells are sufficient to obtain a sample required for downstream applications such as quantitative PCR. Capability to specifically micro-dissect targeted neurons was demonstrated by ∼10-fold increase in mRNA for fluorescent proteins in visually identified neurons expressing the fluorescent proteins compared to neighboring cells not expressing it. We applied this method to validate virus-mediated single-cell knockout, which showed up to 95% reduction in knocked-out gene RNA compared with wild-type neurons. This method using fluorescent proteins fused to transmembrane proteins provides a new, simple solution to perform gene expression analysis in sparsely labelled neuronal/cellular populations, which is especially advantageous when genetic labelling has limited specificity.Significance statementGenetic labeling of specific cell types with reporter fluorescent proteins is widely used for gene expression analysis. However, because the specificity of genetic labeling is often limited, an additional method is required to collect samples from a specific cell type. Here, we developed a novel method to allow for selection of a cell/neuronal type of interest based on morphology before performing sample collection using laser capture microdissection. Stable fluorescent signals from fluorescent proteins fused to transmembrane proteins allows us to morphologically select and laser-capture hundreds of single neurons. This method would be applicable to genomic and transcriptomic analysis of different cell/neuronal types and could facilitate our understanding of brain functions.