Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation.
The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron,...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2019-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0215602 |
| _version_ | 1819114651357544448 |
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| author | Daniel M Kalb Samantha H Adikari Elizabeth Hong-Geller James H Werner |
| author_facet | Daniel M Kalb Samantha H Adikari Elizabeth Hong-Geller James H Werner |
| author_sort | Daniel M Kalb |
| collection | DOAJ |
| description | The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1β (interleukin 1β) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1β and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1β and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response. |
| first_indexed | 2024-12-22T04:48:41Z |
| format | Article |
| id | doaj.art-193aa1b8148345c9b27f8badafd4fdf8 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T04:48:41Z |
| publishDate | 2019-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-193aa1b8148345c9b27f8badafd4fdf82022-12-21T18:38:32ZengPublic Library of Science (PLoS)PLoS ONE1932-62032019-01-01144e021560210.1371/journal.pone.0215602Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation.Daniel M KalbSamantha H AdikariElizabeth Hong-GellerJames H WernerThe heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1β (interleukin 1β) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1β and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1β and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response.https://doi.org/10.1371/journal.pone.0215602 |
| spellingShingle | Daniel M Kalb Samantha H Adikari Elizabeth Hong-Geller James H Werner Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation. PLoS ONE |
| title | Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation. |
| title_full | Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation. |
| title_fullStr | Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation. |
| title_full_unstemmed | Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation. |
| title_short | Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation. |
| title_sort | single cell correlations of mrna and protein content in a human monocytic cell line after lps stimulation |
| url | https://doi.org/10.1371/journal.pone.0215602 |
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