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dc.contributor.authorChang, Ching Chingen_US
dc.contributor.authorChong, Hai Tarngen_US
dc.contributor.authorTashiro, Ayumuen_US
dc.identifier.citationChang, 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-.
dc.description.abstractGene 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.en_US
dc.description.sponsorshipMinistry of Education (MOE)en_US
dc.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.en_US
dc.subjectScience::Biological sciencesen_US
dc.titleLaser capture microdissection of single neurons with morphological visualization using fluorescent proteins fused to transmembrane proteinsen_US
dc.typeJournal Articleen
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.description.versionPublished versionen_US
dc.subject.keywordsGene Expressionen_US
dc.description.acknowledgementThis research was supported by the Ministry of Education, Singapore, Academic Research Fund Tier 1 (Grants 2016-T1-001-010, 2018-T1-002-053) and Tier 3 (Grant MOE2017-T3-1-002).en_US
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