Single-cell RT-PCR identification of genes expressed by human islet endocrine cells.
ABSTRACT 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.