Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling

Abstract

Human beta-cell gene profiling is a powerful tool for understanding beta-cell biology in normal and pathological conditions. The assessment is complicated when isolated islets are studied because of contamination by non-beta-cells and the exposure to the trauma of isolation that causes changes in gene expression. These limitations can be overcome by dissecting the beta-cells from the pancreatic tissue directly using the laser capture microdissection (LCM) technique. LCM allows the sampling of specific cell types from tissue sections. The technique requires morphological criteria or specific stains for targeted cells, and the protocols must preserve the condition of the sought-after macromolecules. We have developed a protocol of rapid tissue dehydration followed by identification of human beta-cells by their intrinsic autofluorescence, which allows laser microdissection for gene-profiling studies.