Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/154247
Title: Laser capture microdissection of single neurons with morphological visualization using fluorescent proteins fused to transmembrane proteins
Authors: Chang, Ching Ching
Chong, Hai Tarng
Tashiro, Ayumu
Keywords: Science::Biological sciences
Issue Date: 2021
Source: Chang, C. C., Chong, H. T. & Tashiro, A. (2021). Laser capture microdissection of single neurons with morphological visualization using fluorescent proteins fused to transmembrane proteins. ENeuro, 8(5), ENEURO.0275-20.2021-. https://dx.doi.org/10.1523/ENEURO.0275-20.2021
Project: 2016-T1-001-010
2018-T1-002-053
MOE2017-T3-1-002 
Journal: eNeuro
Abstract: Gene expression analysis in individual neuronal types helps in understanding 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 labeling 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 the disappearance of fluorescence signals during the tissue processing that is 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 microdissect targeted neurons was demonstrated by an ∼10-fold increase in mRNA for fluorescent proteins in visually identified neurons expressing the fluorescent proteins compared with neighboring cells not expressing it. We applied this method to validate virus-mediated single-cell knockout, which showed up to 92% 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 labeled neuronal/cellular populations, which is especially advantageous when genetic labeling has limited specificity.
URI: https://hdl.handle.net/10356/154247
ISSN: 2373-2822
DOI: 10.1523/ENEURO.0275-20.2021
Rights: © 2021 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Journal Articles

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