Single-Cell RT-PCR Identification of Genes Expressed by Human Islet Endocrine Cells

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.