Assessment of gene expression in head and neck carcinoma using laser capture microdissection and real-time reverse transcription polymerase chain reaction.
OBJECTIVE
To quantify gene expression in tumor cells from human head and neck squamous cell carcinomas (HNSCC) using laser capture microdissection (LCM).


METHODS
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).


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 mum 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.
Malhotra
Pramit S
PS
Department of Otolaryngology-Head and Neck Surgery, University of Minnesota Hospitals, Minneapolis, Minnesota, USA
Malekfzali
Ashi
A
Bonner
Robert F
RF
Juhn
Steven
S
Van Waes
Carter
C
Chen
Zhong
Z
D001707:Biopsy, Needle; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D015331:Cohort Studies; D005260:Female; D005629:Frozen Sections; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008297:Male; D042282:Microdissection; D011336:Probability; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
D012334:RNA, Neoplasm; D042461:Vascular Endothelial Growth Factor A