Gene expression of purified beta-cell tissue obtained from human pancreas with laser capture microdissection

Abstract

Context: Human beta-cell gene profiling is a powerful tool for understanding beta-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-beta-cells and the trauma of the isolation procedure.

Objective: The objective was to use laser capture microdissection (LCM) of human beta-cells from pancreases of cadaver donors and compare their gene expression with that of handpicked isolated islets.

Design: Endogenous autofluorescence of beta-cells facilitated procurement of purified beta-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected beta-cell samples and three isolated islet preparations were obtained. The array data were normalized using DNA-Chip Analyzer software (Harvard School of Public Health, Boston, MA), and the lower confidence bound evaluated differentially expressed genes. Real-time PCR was performed on selected acinar genes and on the duct cell markers, carbonic anhydrase II and keratin 19.

Results: Endogenous autofluorescence facilitates the microdissection of beta-cell rich tissue in human pancreas. When compared with array profiles of purified beta-cell tissue, with lower confidence bound set at 1.2, there were 4560 genes up-regulated and 1226 genes down-regulated in the isolated islets. Among the genes up-regulated in isolated islets were pancreatic acinar and duct genes, chemokine genes, and genes associated with hypoxia, apoptosis, and stress. Quantitative RT-PCR confirmed the differential expression of acinar gene transcripts and the duct marker carbonic anhydrase II in isolated islets.

Conclusion: LCM makes it possible to obtain beta-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.

Figure 1

Flow chart of the experimental design and the procedures used.

Figure 2

Immunofluorescent staining and autofluorescent signal of frozen pancreatic tissue. Insulin or glucagon antibody localization showed by Texas Red (red) and β-cell autofluorescent signal showed by green fluorescence. A and D, Autofluorescent signal in pancreatic islets. B, Pancreatic sections stained with insulin antibody. E, Pancreatic sections stained with glucagon antibody. C, Insulin and autofluorescent signals are colocalized. F, Glucagon signal is distinct from that of autofluorescence. Bar, 100 μm.

Figure 3

Agarose electrophoresis of total and aRNA. Gel electrophoresis was performed to evaluate the integrity of total RNA extracted from isolated islets and the quality of RNA amplified from isolated islets and laser-captured β-cell samples. Total RNA from handpicked isolated islets shows two bands, corresponding to 18S and 28S ribosomal RNA (lane 2). aRNA from isolated islets (lane 3) and microdissected β-cells (lane 4) appear as a smear ranging between 200 and 2000 bases in length. Lane 1, RNA marker.