Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

ABSTRACT: Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment. Samples represent cortex and hippocampus neuron cells collected by pipette and TIVA captured.

ORGANISM(S): Mus musculus

SUBMITTER: Stephen Fisher

PROVIDER: E-GEOD-52525 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Lovatt Ditte D Ruble Brittani K BK Lee Jaehee J Dueck Hannah H Kim Tae Kyung TK Fisher Stephen S Francis Chantal C Spaethling Jennifer M JM Wolf John A JA Grady M Sean MS Ulyanova Alexandra V AV Yeldell Sean B SB Griepenburg Julianne C JC Buckley Peter T PT Kim Junhyong J Sul Jai-Yoon JY Dmochowski Ivan J IJ Eberwine James J

Nature methods 20140112 2

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that th ...[more]

PMID: 24412976

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Project description:Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue’s endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity – with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue’s endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity – with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.

Dataset Information

Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

Publications

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Similar Datasets

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