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

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          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.

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          Most cited references 15

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          Applying the principles of stem-cell biology to cancer.

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            Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.

            Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.
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              MICRODROPLET ASSAY OF HUMAN SERUM CYTOTOXINS.

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                Author and article information

                Contributors
                Journal
                Biol Proced Online
                Biol Proced Online
                Biological Procedures Online
                BioMed Central
                1480-9222
                2014
                8 May 2014
                : 16
                : 9
                Affiliations
                [1 ]Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA
                [2 ]School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
                Article
                1480-9222-16-9
                10.1186/1480-9222-16-9
                4022543
                Copyright © 2014 Yaron et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Categories
                Methodology

                Life sciences

                single cell, rt-qpcr, gene expression analysis, fluorescence microscopy

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