Dataset Information

A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.

ABSTRACT: BACKGROUND:Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. RESULTS:We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. CONCLUSIONS:Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses.

SUBMITTER: Yaron JR

PROVIDER: S-EPMC4022543 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.

Yaron Jordan R JR Ziegler Colleen P CP Tran Thai H TH Glenn Honor L HL Meldrum Deirdre R DR

Biological procedures online 20140508

<h4>Background</h4>Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires special ...[more]

PMID: 24834016

Similar Datasets

Project description:Background:Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell's integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique. Results:Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm's accurate assessment of DNA content was validated by parallel flow cytometric studies. Conclusions:This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

Dataset Information

A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.

Publications

A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets