Summary
Studies of gene expression by different islet endocrine cell populations can provide useful information about signal transduction cascades regulating α-, β- and δ-cell function. Experiments on expression of β-cell gene products are relatively easy to perform in rodent islets as these islets are readily isolated at high purities from the exocrine pancreas; β-cells are the majority cell type and their autofluorescent properties allow them to be purified from non-β-cells by fluorescence-activated cell sorting (FACS). However, the situation is rather more complicated when investigating human islet gene expression profiles as purities of collagenase-isolated human islets are generally less than those of mouse and rat islets; β-cells are less abundant in human islets than they are in rodent islets and conventional FACS purification of human islet β-cells is not possible because of their high background fluorescence. In addition, FACS does not provide pure α- or δ-cell populations from either rodent or human islets. We have developed single-cell RT-PCR protocols to allow identification of genes expressed by human islet α-, β- and δ-cells. This chapter describes these protocols and appropriate steps that should be followed to minimise generation of false-positive amplicons.
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Acknowledgements
The authors are grateful to Dr. S. Pickering for initial advice on appropriate methods for single-cell selection, to Ms. H. Mandefield, the transplantation co-ordinators of South-Thames and King’s College Hospital and relatives of the organ donors for human pancreases, and to Professor S. Amiel and Dr. G.C. Huang for provision of isolated human islets. We thank Rongrong Chen for providing details of nucleotide sequencing. Dr. D. Muller was a Diabetes UK RD Lawrence Research Fellow.
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Muller, D., Jones, P.M., Persaud, S.J. (2009). Single-Cell RT-PCR Identification of Genes Expressed by Human Islet Endocrine Cells. In: Stocker, C. (eds) Type 2 Diabetes. Methods in Molecular Biology, vol 560. Humana Press. https://doi.org/10.1007/978-1-59745-448-3_7
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DOI: https://doi.org/10.1007/978-1-59745-448-3_7
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