Am J Cancer Res 2013;3(4):402-410

Original Article
Validation of esophageal squamous cell carcinoma candidate genes from
high-throughput transcriptomic studies

Qiang Du, Wusheng Yan, Victoria H Burton, Stephen M Hewitt, Lemin Wang, Nan Hu, Philip R Taylor, Michael D Armani, Sumana
Mukherjee, Michael R Emmert-Buck, Michael A Tangrea

Pathogenetics Unit, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA; Tissue Array Research Program, Applied Molecular Pathology Laboratory, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland, USA; Genetic Epidemiology Branch, Division of Cancer Epidemiology and
Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

Received July 11, 2013; Accepted July 30, 2013; Epub August 14, 2013; Published August 30, 2013

Abstract: In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell
carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA
microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and
EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating
protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology
was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of
KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two
relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections
and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four
genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological
information on the overall process of candidate gene validation. (ajcr0000215).

Keywords: Expression microdissection, esophageal squamous cell carcinoma, RNAscope, immunoblot

Address correspondence to: Dr. Michael R Emmert-Buck, Pathogenetics Unit, Laboratory of Pathology, Center for Cancer
Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. E-mail: buckm@mail.nih.gov
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American Journal of Cancer Research
ISSN: 2156-6976