mRNA analysis of intracytoplasmically-stained, FACS-purified pancreatic islet cells using the quantitative nuclease protection assay

Exploring the pathophysiology underlying diabetes mellitus requires characterizing the cellular constituents of pancreatic islets, primarily insulin-producing β-cells. Such efforts have been limited by inadequate techniques for purifying islet cellular subsets for further biochemical and gene-expression studies. Using intracytoplasmic staining and fluorescence-activated cell-sorting (FACS) followed by quantitative nuclease protection assay (qNPA ™ ) technology, we examined 30 relevant genes expressed by islet subpopulations. Purified islet cell subsets expressed all four tested “housekeeping” genes with a surprising variability, dependent on both cell lineage and developmental stage, suggesting caution when interpreting housekeeping gene-normalized mRNA quantifications. Our new approach confirmed expected islet cell lineage-specific gene expression patterns at the transcriptional level, but also detected new phenotypes, including mRNA-profiles (supported by immunohistology) demonstrating that during pregnancy, some β-cells express Mafb, previously found only in immature β-cells during embryonic development. Overall, qNPA ™ gene expression analysis using intracellular-stained then FACS-sorted cells has broad applications beyond islet cell biology.