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High-throughput imaging of mRNA at the single-cell level in human primary immune cells

Measurement of gene expression at the single-cell level has advanced the study of transcriptional regulation programs in healthy and disease states. In particular, single-cell approaches have shed light on the high level of transcriptional heterogeneity of individual cells, both at baseline and in r...

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Autores principales: Gadkari, Manasi, Sun, Jing, Carcamo, Adrian, Alessi, Hugh, Hu, Zonghui, Fraser, Iain D.C., Pegoraro, Gianluca, Franco, Luis M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9380748/
https://www.ncbi.nlm.nih.gov/pubmed/35764396
http://dx.doi.org/10.1261/rna.079239.122
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author Gadkari, Manasi
Sun, Jing
Carcamo, Adrian
Alessi, Hugh
Hu, Zonghui
Fraser, Iain D.C.
Pegoraro, Gianluca
Franco, Luis M.
author_facet Gadkari, Manasi
Sun, Jing
Carcamo, Adrian
Alessi, Hugh
Hu, Zonghui
Fraser, Iain D.C.
Pegoraro, Gianluca
Franco, Luis M.
author_sort Gadkari, Manasi
collection PubMed
description Measurement of gene expression at the single-cell level has advanced the study of transcriptional regulation programs in healthy and disease states. In particular, single-cell approaches have shed light on the high level of transcriptional heterogeneity of individual cells, both at baseline and in response to experimental or environmental perturbations. We have developed a method for high-content imaging (HCI)-based quantification of relative changes in transcript abundance at the single-cell level in human primary immune cells and have validated its performance under multiple experimental conditions to demonstrate its general applicability. This method, named hcHCR, combines the sensitivity of the hybridization chain reaction (HCR) for the visualization of RNA in single cells, with the speed, scalability, and reproducibility of HCI. We first tested eight cell attachment substrates for short-term culture of primary human B cells, T cells, monocytes, or neutrophils. We then miniaturized HCR in 384-well format and documented the ability of the method to detect changes in transcript abundance at the single-cell level in thousands of cells for each experimental condition by HCI. Furthermore, we demonstrated the feasibility of multiplexing gene expression measurements by simultaneously assaying the abundance of three transcripts per cell at baseline and in response to an experimental stimulus. Finally, we tested the robustness of the assay to technical and biological variation. We anticipate that hcHCR will be suitable for low- to medium-throughput chemical or functional genomics screens in primary human cells, with the possibility of performing screens on cells obtained from patients with a specific disease.
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spelling pubmed-93807482023-09-01 High-throughput imaging of mRNA at the single-cell level in human primary immune cells Gadkari, Manasi Sun, Jing Carcamo, Adrian Alessi, Hugh Hu, Zonghui Fraser, Iain D.C. Pegoraro, Gianluca Franco, Luis M. RNA Method Measurement of gene expression at the single-cell level has advanced the study of transcriptional regulation programs in healthy and disease states. In particular, single-cell approaches have shed light on the high level of transcriptional heterogeneity of individual cells, both at baseline and in response to experimental or environmental perturbations. We have developed a method for high-content imaging (HCI)-based quantification of relative changes in transcript abundance at the single-cell level in human primary immune cells and have validated its performance under multiple experimental conditions to demonstrate its general applicability. This method, named hcHCR, combines the sensitivity of the hybridization chain reaction (HCR) for the visualization of RNA in single cells, with the speed, scalability, and reproducibility of HCI. We first tested eight cell attachment substrates for short-term culture of primary human B cells, T cells, monocytes, or neutrophils. We then miniaturized HCR in 384-well format and documented the ability of the method to detect changes in transcript abundance at the single-cell level in thousands of cells for each experimental condition by HCI. Furthermore, we demonstrated the feasibility of multiplexing gene expression measurements by simultaneously assaying the abundance of three transcripts per cell at baseline and in response to an experimental stimulus. Finally, we tested the robustness of the assay to technical and biological variation. We anticipate that hcHCR will be suitable for low- to medium-throughput chemical or functional genomics screens in primary human cells, with the possibility of performing screens on cells obtained from patients with a specific disease. Cold Spring Harbor Laboratory Press 2022-09 /pmc/articles/PMC9380748/ /pubmed/35764396 http://dx.doi.org/10.1261/rna.079239.122 Text en Published by Cold Spring Harbor Laboratory Press for the RNA Society https://creativecommons.org/licenses/by-nc/4.0/This is a work of the US government.
spellingShingle Method
Gadkari, Manasi
Sun, Jing
Carcamo, Adrian
Alessi, Hugh
Hu, Zonghui
Fraser, Iain D.C.
Pegoraro, Gianluca
Franco, Luis M.
High-throughput imaging of mRNA at the single-cell level in human primary immune cells
title High-throughput imaging of mRNA at the single-cell level in human primary immune cells
title_full High-throughput imaging of mRNA at the single-cell level in human primary immune cells
title_fullStr High-throughput imaging of mRNA at the single-cell level in human primary immune cells
title_full_unstemmed High-throughput imaging of mRNA at the single-cell level in human primary immune cells
title_short High-throughput imaging of mRNA at the single-cell level in human primary immune cells
title_sort high-throughput imaging of mrna at the single-cell level in human primary immune cells
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9380748/
https://www.ncbi.nlm.nih.gov/pubmed/35764396
http://dx.doi.org/10.1261/rna.079239.122
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