Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies
Open accessAbstract
Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.
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Immediate early gene expression in sorted microglia is an experimental artifact; adult microglia in tissue are negative for Fos and Egr1.
“Indeed, adult microglia stained negative for Fos/Egr1 transcripts or proteins, whereas their expression was readily detected in the surrounding non-microglial cells”
From Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing (2019) · cites this paper as Haimon et al., 2018
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References 31 · Cited by 209
Research works printed but not linked: 5
- [1]Yona S Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis Immunity 2013 38 79 91 23273845 PMC3908543
- [2]Sanz E Cell-type-specific isolation of ribosome-associated mRNA from complex tissues Proceedings of the National Academy of Sciences 2009 106 13939 13944 PMC2728999 19666516
- [4]Lavin Y Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local Microenvironment Cell 2014 159 1312 1326 25480296 PMC4437213
- [8]Gosselin D Environment Drives Selection and Function of Enhancers Controlling Tissue-Specific Macrophage Identities Cell 2014 159 1327 1340 25480297 PMC4364385
- [9]Halpern KB Nuclear Retention of mRNA in Mammalian Tissues CellReports 2015 13 2653 2662 PMC4700052 26711333
- Authors
- Haimon, Z., Volaski, A., Orthgiess, J., Boura-Halfon, S., Varol, D., Shemer, A., Yona, S., Zuckerman, B., David, E., Chappell-Maor, L., Bechmann, I., Gericke, M., Ulitsky, I., & Jung, S.
- Affiliations
- Weizmann Institute of Science (IL)
- Leipzig University (DE)
- Corresponding author
- Jung, Steffen
- ORCID
- Venue
- Nature Immunology
- Published
- 2018-05-18
- Volume
- 19
- Issue
- 6
- Pages
- 636-644
- ISSN
- 1529-2908 (ISSN-L)
- 1529-2916
- PMC
- PMC5986066
- OpenAlex
- W2804756252
- Cited by
- 209
- References
- 31
- Citations by year
- 2026: 12 · 2025: 11 · 2024: 26 · 2023: 24 · 2022: 27 · 2021: 34 · 2020: 40 · 2019: 28 · 2018: 7
- FWCI
- 9.69
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Immunology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- UCL Discovery (University College London) (submittedVersion)
- Funders
- Deutsche Forschungsgemeinschaft
- Israel Science Foundation
- Nature
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- microglia, RiboTag, tissue dissociation, transgenic mouse model, gene expression profiling
- MeSH terms
- Animals, Mice, Inbred C57BL, Mice, Transgenic, Ribosomes, RNA, Messenger, Sequence Analysis, RNA, Microglia, Immunoprecipitation, Mice
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