Abstract
Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet–cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.
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Acknowledgements
The authors like to thank A. Franks for mouse colony management and S. Wank, O. Gavrilova and L. Weinstein for critically reading the manuscript and making helpful suggestions. This research was supported in part by the Intramural Research Program of the US National Institutes of Health (NIH), National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK), and NIH DK042502, Juvenile Diabetes Research Foundation 1-2008-595 to R.S.
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Seligman is the founder and CSO of High Throughput Genomics. Seligman owns stock in High Throughput Genomics, the company that developed and markets the qNPA assay, services, kits and related imagers used to measure signal from those kits.
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Pechhold, S., Stouffer, M., Walker, G. et al. Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection. Nat Biotechnol 27, 1038–1042 (2009). https://doi.org/10.1038/nbt.1579
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DOI: https://doi.org/10.1038/nbt.1579
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