There are a variety of options for making microarrays and obtaining microarray data. Here, we describe the building and use of two microarray facilities in academic settings. In addition to specifying technical detail, we comment on the advantages and disadvantages of components and approaches, and provide a protocol for hybridization. The fact that we are now making and using microarrays to answer biological questions demonstrates that the technology can be implemented in a university environment.

Small noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. The importance of miRNAs as potential cancer prognostic indicators is underscored by their involvement in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) tumor and adjacent normal tissue were examined by microarray analysis and validated by quantitative TaqMan real-time polymerase chain reaction. Using TaqMan real-time polymerase chain reaction we measured the quantitative associations between a subset of miRNAs identified on microarrays in primary tumors at diagnosis and cancer survival in a cohort of 104 HNSCC patients undergoing treatment with curative intent. The majority of miRNAs exhibiting altered expression in primary human HNSCC tumors (including miR-1, miR-133a, miR-205, and let7d) show lower expression levels relative to normal adjacent tissue. In contrast, hsa-miR-21 is frequently overexpressed in human HNSCC tumors. Using univariate and multivariable statistical models we show that low levels of hsa-miR205 are significantly associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. In addition , combined low levels of hsa-miR-205 and hsa-let-7d expression in HNSCC tumors are significantly associated with poor head and neck cancer survival Our results show that miRNA expression levels can be used as prognostic markers of head and neck cancer.

Purpose: We have utilized a genome-wide approach to identify novel differentially methylated CpG dinucleotides that are seen in different anatomic sites of head and neck squamous cell carcinoma (HNSCC), as well as those that might be related to HPV status in the oropharynx. Experimental Design: We performed genome-wide DNA methylation profiling of primary tumor samples and corresponding adjacent mucosa from 118 HNSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M-value). Results: When datasets were individually analyzed by anatomic site of the primary tumor, we identified 293 differentially methylated CpG loci in oral cavity SCC, 219 differentially methylated CpG loci in laryngeal SCC, and 460 differentially methylated in oropharyngeal SCC. A subset of these differentially methylated CpG loci was common across all anatomic sites of HNSCC. Stratification by HPV status revealed a significantly higher number of differentially methylated CpG loci in HPV-positive patients. Conclusion: Novel epigenetic biomarkers derived from clinical HNSCC specimens can be used molecular classifiers of this disease, revealing many new avenues of investigation for this disease.