An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells

Abstract Exploring the variability in gene expressions of rare cells at the single-cell level is critical for understanding mechanisms of differentiation in tissue function and development as well as for disease diagnostics and cancer treatment. Such studies, however, have been hindered by major dif...

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Autores principales:	Junyi Shang, David Welch, Manuela Buonanno, Brian Ponnaiya, Guy Garty, Timothy Olsen, Sally A. Amundson, Qiao Lin
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2019
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/725de23da98e468dbcff9ad8f04f47dd
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spelling	oai:doaj.org-article:725de23da98e468dbcff9ad8f04f47dd2021-12-02T13:35:12ZAn Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells10.1038/s41598-019-55831-22045-2322https://doaj.org/article/725de23da98e468dbcff9ad8f04f47dd2019-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-019-55831-2https://doaj.org/toc/2045-2322Abstract Exploring the variability in gene expressions of rare cells at the single-cell level is critical for understanding mechanisms of differentiation in tissue function and development as well as for disease diagnostics and cancer treatment. Such studies, however, have been hindered by major difficulties in tracking the identity of individual cells. We present an approach that combines single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis of rare cells. The approach utilizes a photocleavage bead-based microfluidic device to synthesize and deliver stable cDNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination. The utility of the approach was demonstrated with QuantStudio digital PCR by analyzing the radiation and bystander effect on individual IMR90 human lung fibroblasts. Expression levels of the Cyclin-dependent kinase inhibitor 1a (CDKN1A), Growth/differentiation factor 15 (GDF15), and Prostaglandin-endoperoxide synthase 2 (PTGS2) genes, previously shown to have different responses to direct and bystander irradiation, were measured across individual control, microbeam-irradiated or bystander IMR90 cells. In addition to the confirmation of accurate tracking of cell treatments through the system and efficient analysis of single-cell responses, the results enable comparison of activation levels of different genes and provide insight into signaling pathways within individual cells.Junyi ShangDavid WelchManuela BuonannoBrian PonnaiyaGuy GartyTimothy OlsenSally A. AmundsonQiao LinNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 9, Iss 1, Pp 1-12 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Junyi Shang David Welch Manuela Buonanno Brian Ponnaiya Guy Garty Timothy Olsen Sally A. Amundson Qiao Lin An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells
description	Abstract Exploring the variability in gene expressions of rare cells at the single-cell level is critical for understanding mechanisms of differentiation in tissue function and development as well as for disease diagnostics and cancer treatment. Such studies, however, have been hindered by major difficulties in tracking the identity of individual cells. We present an approach that combines single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis of rare cells. The approach utilizes a photocleavage bead-based microfluidic device to synthesize and deliver stable cDNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination. The utility of the approach was demonstrated with QuantStudio digital PCR by analyzing the radiation and bystander effect on individual IMR90 human lung fibroblasts. Expression levels of the Cyclin-dependent kinase inhibitor 1a (CDKN1A), Growth/differentiation factor 15 (GDF15), and Prostaglandin-endoperoxide synthase 2 (PTGS2) genes, previously shown to have different responses to direct and bystander irradiation, were measured across individual control, microbeam-irradiated or bystander IMR90 cells. In addition to the confirmation of accurate tracking of cell treatments through the system and efficient analysis of single-cell responses, the results enable comparison of activation levels of different genes and provide insight into signaling pathways within individual cells.
format	article
author	Junyi Shang David Welch Manuela Buonanno Brian Ponnaiya Guy Garty Timothy Olsen Sally A. Amundson Qiao Lin
author_facet	Junyi Shang David Welch Manuela Buonanno Brian Ponnaiya Guy Garty Timothy Olsen Sally A. Amundson Qiao Lin
author_sort	Junyi Shang
title	An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells
title_short	An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells
title_full	An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells
title_fullStr	An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells
title_full_unstemmed	An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells
title_sort	integrated preprocessing approach for exploring single-cell gene expression in rare cells
publisher	Nature Portfolio
publishDate	2019
url	https://doaj.org/article/725de23da98e468dbcff9ad8f04f47dd
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An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells

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