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 alpha-, beta- and delta-cell function. Experiments on expression of beta-cell gene products are relatively easy to perform in rodent islets as these islets are readily isolated at high purities from the exocrine pancreas; beta-cells are the majority cell type and their autofluorescent properties allow them to be purified from non-beta-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; beta-cells are less abundant in human islets than they are in rodent islets and conventional FACS purification of human islet beta-cells is not possible because of their high background fluorescence. In addition, FACS does not provide pure alpha- or delta-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 alpha-, beta- and delta-cells. This chapter describes these protocols and appropriate steps that should be followed to minimise generation of false-positive amplicons.