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...

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Bibliographic Details
Published in:	PloS one Vol. 18; no. 8; p. e0289279
Main Authors:	Stanley, John, Lohith, Akshar, Debiaso, Lucca, Wang, Kevan, Ton, Minh, Cui, Wenwu, Gu, Weiwei, Fu, Aihua, Pourmand, Nader
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 01-08-2023
Subjects:	Analysis Automation Bar codes Biology and life sciences Breast cancer Cell division Cells Data acquisition Dissociation Engineering and Technology Enzymes Genomics Hybridization Microscopy Nanoparticles Physical Sciences Research and analysis methods Ribonucleic acid RNA RNA sequencing Technology Tissues Transcriptomics United States
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0289279