Frozen biopsies were embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA) for serial 6µm cryosectioning. Step-sections were stained with hematoxylin and eosin (H&E) and intervening unstained sections were evaluated for cytokeratins using monoclonal mouse anti-human cytokeratin antibody (AE1/AE3, 1:20 dilution) and Envision+ System Peroxidase (DAB) (DAKO Corporation, Carpinteria, CA). EBV-harboring cells were identified by detecting EBV-encoded small RNAs (EBERs) using the EBV Probe In Situ Hybridization Kit (NCL-EBV-K, Novocastra laboratories, Newcastle upon Tyne, UK). Laser capture microdissection (LCM) was performed on tumor samples containing < 70% tumor cells, and to dissect epithelial cells from normal healthy nasopharyngeal specimens, using an Arcturus Pixcell-II LCM microscope and CapSure Macro LCM caps (Arcturus Bioscience, Mt. View, CA). Typically, from one to a few thousand cells per sample were captured.
Growth protocol
Tumor and healthy nasopharyngeal tissue from areas macroscopically not involved in the tumor, was collected with informed consent under approval of the Human Subjects Institutional Review Boards of National Taiwan University, UW-Madison, and U.S. NCI. After resection, specimens were immediately flash frozen and stored in liquid nitrogen. Histopathology and tumor staging definitions and information are provided in Sengupta et al, 2006, Cancer Research 66(16): 7999-8006.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Trizol (Invitrogen, Carlsbad, CA), and DNaseI treated.
Label
biotin
Label protocol
Polyadenylated RNA was amplified twice using Affymetrix’s Small Sample Labeling Protocol VII. One-twentieth of second round cDNA was used to assay efficiency of amplification by measuring β-actin using the Quantitect SYBR Green real time PCR kit (Qiagen, Valencia, CA). Using the BioArray High Yield RNA Labeling Kit (ENZO Life Sciences, Farmingdale, NY), half of the second round cDNA was used to synthesize biotinylated antisense RNA (aRNA).
Hybridization protocol
Fragmented, biotinylated aRNA was hybridized to Affymetrix HG U133 Plus 2.0 microarrays, as per Affymetrix-recommended procedures.
Scan protocol
Microarray scanning and data acquisition was carried out at the University of Wisconsin-Madison Gene Expression Center using Affymetrix-recommended equipment and procedures.
Description
Total RNA extracted from laser-captured epithelium from 31 nasopharyngeal carcinomas and 10 normal healthy nasopharyngeal tissue specimens.
Data processing
Detailed description of data analyses and statistical methods used are provided in two primary publications: Sengupta et al, 2006, Cancer Research 66(16): 7999-8006; Dodd et al, 2006, Cancer Epidemiology, Biomarkers & Prevention 15(11): 2216-2225.
