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High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality
Single-cell transcriptomics is essential for understanding biological variability among cells in a heterogenous population. Acquiring high-quality single-cell sequencing data from a tissue sample has multiple challenges including isolation of individual cells as well as amplification of the genetic...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10393160/ https://www.ncbi.nlm.nih.gov/pubmed/37527243 http://dx.doi.org/10.1371/journal.pone.0289279 |
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author | Stanley, John Lohith, Akshar Debiaso, Lucca Wang, Kevan Ton, Minh Cui, Wenwu Gu, Weiwei Fu, Aihua Pourmand, Nader |
author_facet | Stanley, John Lohith, Akshar Debiaso, Lucca Wang, Kevan Ton, Minh Cui, Wenwu Gu, Weiwei Fu, Aihua Pourmand, Nader |
author_sort | Stanley, John |
collection | PubMed |
description | Single-cell transcriptomics is essential for understanding biological variability among cells in a heterogenous population. Acquiring high-quality single-cell sequencing data from a tissue sample has multiple challenges including isolation of individual cells as well as amplification of the genetic material. Commercially available techniques require the isolation of individual cells from a tissue through extensive manual manipulation before single cell sequence data can be acquired. However, since cells within a tissue have different dissociation constants, enzymatic and mechanical manipulation do not guarantee the isolation of a homogenous population of cells. To overcome this drawback, in this research we have developed a revolutionary approach that utilizes a fully automated nanopipette technology in combination with magnetic nanoparticles to obtain high quality sequencing reads from individual cells within an intact tissue thereby eliminating the need for manual manipulation and single cell isolation. With the proposed technology, it is possible to sample an individual cell within the tissue multiple times to obtain longitudinal information. Single-cell RNAseq was achieved by aspirating only1-5% of sub-single-cell RNA content from individual cells within fresh frozen tissue samples. As a proof of concept, aspiration was carried out from 22 cells within a breast cancer tissue slice using quartz nanopipettes. The mRNA from the aspirate was then selectively captured using magnetic nanoparticles. The RNAseq data from aspiration of 22 individual cells provided high alignment rates (80%) with 2 control tissue samples. The technology is exceptionally simple, quick and efficient as the entire cell targeting and aspiration process is fully automated. |
format | Online Article Text |
id | pubmed-10393160 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-103931602023-08-02 High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality Stanley, John Lohith, Akshar Debiaso, Lucca Wang, Kevan Ton, Minh Cui, Wenwu Gu, Weiwei Fu, Aihua Pourmand, Nader PLoS One Research Article Single-cell transcriptomics is essential for understanding biological variability among cells in a heterogenous population. Acquiring high-quality single-cell sequencing data from a tissue sample has multiple challenges including isolation of individual cells as well as amplification of the genetic material. Commercially available techniques require the isolation of individual cells from a tissue through extensive manual manipulation before single cell sequence data can be acquired. However, since cells within a tissue have different dissociation constants, enzymatic and mechanical manipulation do not guarantee the isolation of a homogenous population of cells. To overcome this drawback, in this research we have developed a revolutionary approach that utilizes a fully automated nanopipette technology in combination with magnetic nanoparticles to obtain high quality sequencing reads from individual cells within an intact tissue thereby eliminating the need for manual manipulation and single cell isolation. With the proposed technology, it is possible to sample an individual cell within the tissue multiple times to obtain longitudinal information. Single-cell RNAseq was achieved by aspirating only1-5% of sub-single-cell RNA content from individual cells within fresh frozen tissue samples. As a proof of concept, aspiration was carried out from 22 cells within a breast cancer tissue slice using quartz nanopipettes. The mRNA from the aspirate was then selectively captured using magnetic nanoparticles. The RNAseq data from aspiration of 22 individual cells provided high alignment rates (80%) with 2 control tissue samples. The technology is exceptionally simple, quick and efficient as the entire cell targeting and aspiration process is fully automated. Public Library of Science 2023-08-01 /pmc/articles/PMC10393160/ /pubmed/37527243 http://dx.doi.org/10.1371/journal.pone.0289279 Text en © 2023 Stanley et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Stanley, John Lohith, Akshar Debiaso, Lucca Wang, Kevan Ton, Minh Cui, Wenwu Gu, Weiwei Fu, Aihua Pourmand, Nader High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality |
title | High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality |
title_full | High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality |
title_fullStr | High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality |
title_full_unstemmed | High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality |
title_short | High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality |
title_sort | high throughput isolation of rna from single-cells within an intact tissue for spatial and temporal sequencing a reality |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10393160/ https://www.ncbi.nlm.nih.gov/pubmed/37527243 http://dx.doi.org/10.1371/journal.pone.0289279 |
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