Gene expression profiling of microdissected pancreatic ductal carcinomas using high-density DNA microarrays - PubMed (original) (raw)

. 2004 Sep-Oct;6(5):611-22.

doi: 10.1593/neo.04295.

Christian Pilarsky, Ole Ammerpohl, Jutta Lüttges, Armin Böhme, Bence Sipos, Melanie Foerder, Ingo Alldinger, Beatrix Jahnke, Hans Konrad Schackert, Holger Kalthoff, Bernd Kremer, Günter Klöppel, Hans Detlev Saeger

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Gene expression profiling of microdissected pancreatic ductal carcinomas using high-density DNA microarrays

Robert Grützmann et al. Neoplasia. 2004 Sep-Oct.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of malignancy-related death and is the eighth most common cancer with the lowest overall 5-year relative survival rate. To identify new molecular markers and candidates for new therapeutic regimens, we investigated the gene expression profile of microdissected cells from 11 normal pancreatic ducts, 14 samples of PDAC, and 4 well-characterized pancreatic cancer cell lines using the Affymetrix U133 GeneChip set. RNA was extracted from microdissected samples and cell lines, amplified, and labeled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. We found 616 differentially expressed genes. Within these, 140 were also identified in PDAC by others, such as Galectin-1, Galectin-3, and MT-SP2. We validated the differential expression of several genes (e.g., CENPF, MCM2, MCM7, RAMP, IRAK1, and PTTG1) in PDAC by immunohistochemistry and reverse transcription polymerase chain reaction. We present a whole genome expression study of microdissected tissues from PDAC, from microdissected normal ductal pancreatic cells and pancreatic cancer cell lines using high-density microarrays. Within the panel of genes, we identified novel differentially expressed genes, which have not been associated with the pathogenesis of PDAC before.

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Figures

Figure 1

Figure 1

Manual microdissection of pancreatic tissue. Left: Before microdissection; right: after microdissection; upper panel: pancreatic ductal adenocarcinoma; lower panel: normal ductal epithelia.

Figure 2

Figure 2

Analysis of gene expression in PDAC. (A) Hierarchical clustering of 14 microdissected PDACs, 11 microdissected normal ductal cells, and 4 established pancreatic tumor cell lines using the 616 differential gene set and a Euclidian distance matrix. The different colors reflect the predominant expression of the genes (green: normal tissue; blue: cell lines and PDAC; red: PDAC). The individual samples are colored (green: normal; blue: cell lines; red: PDAC). (B) Heat map of signature genes in PDAC. Genes were identified using the KNN method with a LOO validation step. (C) Heat map of protein kinase expression found in the set of differentially expressed genes. Genes that are upregulated appear in red, and those that are downregulated appear in green, with the expression value reflected by the intensity of the color.

Figure 3

Figure 3

Immunohistochemical analysis of differentially expressed genes found in PDAC by microarray analysis. CENPF (A–C), MCM7 (D–F), and MCM2 (G–I) are highly expressed in the nucleus of PDACs of different grades (D: G1; A and E: G2; B, C, and F: G3; C, F, and I: normal pancreas). Original magnification, x100 (A, D, and E). Original magnification, x200 (C). Original magnification, x400 (B and F).

Figure 4

Figure 4

Validation of differentially expressed genes using RT-PCR. (A) RT-PCR analysis of five genes. RNA from tumor (T) and corresponding normal tissue (N) from four patients with a PDAC was isolated, and RT-PCR analysis for G6PD (control), PLAG1, PTTG, osf-2, and RAMP1 was performed. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (B) Overview of RT-PCR results from normal pancreatic tissue, PDAC (upper panel), and cell lines (lower panel). GAPDH and G6PD expression analysis served as control. White background: not determined; green: no PCR signal (-), cT value > 35; yellow: PCR signal visible (-/+); orange: clear PCR signal (+), cT value > 27< 35; red: strong PCR signal (++), cT value > 20 < 27; dark red: very strong RT-PCR signal (+++), cT value < 20.

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