Protocol

Type 2 Diabetes

Volume 560 of the series Methods in Molecular Biology pp 87-98

Date:

Laser Capture Microdissection of Human Pancreatic β-Cells and RNA Preparation for Gene Expression Profiling

  • Lorella MarselliAffiliated withSection on Islet Transplantation and Cell Biology, Joslin Diabetes Center
  • , Dennis C. SgroiAffiliated withSection on Islet Transplantation and Cell Biology, Joslin Diabetes Center
  • , Susan Bonner-WeirAffiliated withSection on Islet Transplantation and Cell Biology, Joslin Diabetes Center
  • , Gordon C. WeirAffiliated withSection on Islet Transplantation and Cell Biology, Joslin Diabetes Center Email author 

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Summary

Human β-cell gene profiling is a powerful tool for understanding β-cell biology in normal and pathological conditions. The assessment is complicated when isolated islets are studied because of contamination by non-β-cells and the exposure to the trauma of isolation that causes changes in gene expression. These limitations can be overcome by dissecting the β-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 β-cells by their intrinsic autofluorescence, which allows laser microdissection for gene-profiling studies.

Key words

Human β-cell Laser capture microdissection RNA extraction RNA integrity Type 2 diabetes