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.
Muller
Dany
D
Beta Cell Development & Function Group, King's College London, Guy's Campus, 2.9N Hodgkin Building, London, SE1 1UL, UK.
Jones
Peter M
PM
Persaud
Shanta J
SJ
D003584:Cytological Techniques; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014021:Tissue Preservation