Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Islet-cell gene transcription analysis by qNPA is quantitative and not influenced by cell fixation.
Figure 2: Dissection, purification and gene-expression analysis of islet-cell subsets by FACS-sorting and qNPA.
Figure 3: Facultative Mafb expression in adult mouse beta cells.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Brissova, M. et al. Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J. Histochem. Cytochem. 53, 1087–1097 (2005).

    Article  CAS  Google Scholar 

  2. Nadal, A., Quesada, I. & Soria, B. Homologous and heterologous asynchronicity between identified alpha-, beta- and delta-cells within intact islets of Langerhans in the mouse. J. Physiol. (Lond.) 517, 85–93 (1999).

    Article  CAS  Google Scholar 

  3. Ahn, Y.B. et al. Changes in gene expression in beta cells after islet isolation and transplantation using laser-capture microdissection. Diabetologia 50, 334–342 (2007).

    Article  CAS  Google Scholar 

  4. Pechhold, K. et al. Dynamic changes in pancreatic endocrine cell abundance, distribution, and function in antigen-induced and spontaneous autoimmune diabetes. Diabetes 58, 1175–1184 (2009).

    Article  CAS  Google Scholar 

  5. Pechhold, K. et al. Blood glucose levels regulate pancreatic beta-cell proliferation during experimentally-induced and spontaneous autoimmune diabetes in mice. PLoS ONE 4, e4827 (2009).

    Article  Google Scholar 

  6. Tornehave, D., Kristensen, P., Romer, J., Knudsen, L.B. & Heller, R.S. Expression of the GLP-1 receptor in mouse, rat, and human pancreas. J. Histochem. Cytochem. 56, 841–851 (2008).

    Article  CAS  Google Scholar 

  7. Gu, G. et al. Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development 131, 165–179 (2004).

    Article  CAS  Google Scholar 

  8. Mellitzer, G. et al. Pancreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock-add-on mice. Mol. Endocrinol. 18, 2765–2776 (2004).

    Article  CAS  Google Scholar 

  9. Linden, E., Skoglund, P. & Rundquist, I. Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation. Cytometry 27, 92–95 (1997).

    Article  CAS  Google Scholar 

  10. Sambrook, J. & Russel, D.W. in Molecular Cloning: A Laboratory Manual, edn. 3 (eds. Sambrook J. & Russel D.W.) Chapter 7: Extraction, purification, and analysis of mRNA from eukaryotic cells, 51–62 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

  11. Calzone, F.J., Britten, R.J. & Davidson, E.H. Mapping of gene transcripts by nuclease protection assays and cDNA primer extension. Methods Enzymol. 152, 611–632 (1987).

    Article  CAS  Google Scholar 

  12. Martel, R.R. et al. Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection. Assay Drug Dev. Technol. 1, 61–71 (2002).

    Article  CAS  Google Scholar 

  13. Wersto, R.P. et al. Doublet discrimination in DNA cell-cycle analysis. Cytometry 46, 296–306 (2001).

    Article  CAS  Google Scholar 

  14. de Jonge, H.J. et al. Evidence based selection of housekeeping genes. PLoS ONE 2, e898 (2007).

    Article  Google Scholar 

  15. Frericks, M. & Esser, C. A toolbox of novel murine house-keeping genes identified by meta-analysis of large scale gene expression profiles. Biochim. Biophys. Acta 1779, 830–837 (2008).

    Article  CAS  Google Scholar 

  16. Lee, P.D., Sladek, R., Greenwood, C.M. & Hudson, T.J. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res. 12, 292–297 (2002).

    Article  Google Scholar 

  17. Wang, J., Webb, G., Cao, Y. & Steiner, D.F. Contrasting patterns of expression of transcription factors in pancreatic alpha and beta cells. Proc. Natl. Acad. Sci. USA 100, 12660–12665 (2003).

    Article  CAS  Google Scholar 

  18. Neerman-Arbez, M., Cirulli, V. & Halban, P.A. Levels of the conversion endoproteases PC1 (PC3) and PC2 distinguish between insulin-producing pancreatic islet beta cells and non-beta cells. Biochem. J. 300, 57–61 (1994).

    Article  CAS  Google Scholar 

  19. Marcinkiewicz, M., Ramla, D., Seidah, N.G. & Chretien, M. Developmental expression of the prohormone convertases PC1 and PC2 in mouse pancreatic islets. Endocrinology 135, 1651–1660 (1994).

    Article  CAS  Google Scholar 

  20. Murtaugh, L.C. Pancreas and beta-cell development: from the actual to the possible. Development 134, 427–438 (2007).

    Article  CAS  Google Scholar 

  21. Oliver-Krasinski, J.M. & Stoffers, D.A. On the origin of the beta cell. Genes Dev. 22, 1998–2021 (2008).

    Article  CAS  Google Scholar 

  22. Nishimura, W. et al. A switch from MafB to MafA expression accompanies differentiation to pancreatic beta-cells. Dev. Biol. 293, 526–539 (2006).

    Article  CAS  Google Scholar 

  23. Chimienti, F. et al. In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion. J. Cell Sci. 119, 4199–4206 (2006).

    Article  CAS  Google Scholar 

  24. Gyulkhandanyan, A.V. et al. Investigation of transport mechanisms and regulation of intracellular Zn2+ in pancreatic alpha-cells. J. Biol. Chem. 283, 10184–10197 (2008).

    Article  CAS  Google Scholar 

  25. St Onge, L., Sosa-Pineda, B., Chowdhury, K., Mansouri, A. & Gruss, P. Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. Nature 387, 406–409 (1997).

    Article  CAS  Google Scholar 

  26. Kataoka, K. Multiple mechanisms and functions of maf transcription factors in the regulation of tissue-specific genes. J. Biochem. 141, 775–781 (2007).

    Article  CAS  Google Scholar 

  27. Olbrot, M., Rud, J., Moss, L.G. & Sharma, A. Identification of beta-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA. Proc. Natl. Acad. Sci. USA 99, 6737–6742 (2002).

    Article  CAS  Google Scholar 

  28. Matsuoka, T.A. et al. The MafA transcription factor appears to be responsible for tissue-specific expression of insulin. Proc. Natl. Acad. Sci. USA 101, 2930–2933 (2004).

    Article  CAS  Google Scholar 

  29. Artner, I. et al. MafB: an activator of the glucagon gene expressed in developing islet alpha- and beta-cells. Diabetes 55, 297–304 (2006).

    Article  CAS  Google Scholar 

  30. Parsons, J.A., Brelje, T.C. & Sorenson, R.L. Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion. Endocrinology 130, 1459–1466 (1992).

    CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Klaus Pechhold.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–3 and Supplementary Tables 1–4 (PDF 313 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1579

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing