Assessment of gene expression in head and neck carcinoma using laser capture microdissection and real-time reverse transcription polymerase chain reaction

Pramit S. Malhotra, Ashi Malekfzali, Robert F. Bonner, Steven Juhn, Carter Van Waes, Zhong Chen

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Objectives: To quantify gene expression in tumor cells from human head and neck squamous cell carcinomas (HNSCC) using laser capture microdissection (LCM). Study Design: Histopathologically identified HNSCC cells were microdissected from frozen sections, RNA was isolated, and vascular endothelial growth factor (VEGF) gene expression was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Materials and Methods: Two human HNSCC tumor samples and matched normal mucosal biopsies and five human xenograft tumor specimens were harvested, embedded, and frozen in OCT. The frozen tumors were sectioned to 8 to 10 μm in thickness, and hematoxylin-eosin (H&E) staining was performed before LCM. An estimated 2,000 to 3,000 tumor cells were microdissected from frozen sections and processed for RNA isolation. mRNA for VEGF was analyzed by real time RT-PCR (TaqMan) with commercially available primers and probes. Results: Two thousand to 3000 cells were necessary to obtain a suitable quantity of RNA for subsequent gene expression study by real-time RT-PCR. The gene expression of VEGF, a major tumor angiogenic factor, was tested in microdissected HNSCC and compared with uninvolved normal mucosal controls. A greater than seven-fold increase of VEGF expression in tumor specimens versus mucosal controls was observed. Conclusions: LCM is a novel sample conserving technique that allows the precise selection of tumor cells from a heterogeneous architecture. The combination of LCM and real-time RT-PCR appears particularly efficacious for studying HNSCC molecular pathogenesis and identifying tissue-specific biomarkers.

Original languageEnglish (US)
Pages (from-to)2123-2128
Number of pages6
JournalLaryngoscope
Volume114
Issue number12
DOIs
StatePublished - Dec 2004

Keywords

  • Gene expression
  • Head and neck cancer
  • Laser capture microdissection
  • Real time RT-PCR
  • VEGF

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